genomic hcv rna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher genomic hcv rna
    Disruption in the 5′UTR nt95–110:NS5B nt8528–8543 duplex reduces intracellular <t>HCV</t> <t>RNA</t> levels. ( A ) Huh-7.5 cells transfected with HCV genomic RNA (WT) or HCV genomic RNA containing mutations at 5′UTR nt 95–110 and NS5B nt 8528–8543 express NS5B antigen 24 h post-transfection. Scale bar = 50 µm. ( B ) Plots represent the average number of HCV RNA genome copies ± SE in 1 μg of cellular RNA at 48 h post-transfection. p -values ≤ 0.05 (*) or ≤ 0.01 (**) were determined by the Student’s t -test and represent four or more independent experiments.
    Genomic Hcv Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 15129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5′UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication"

    Article Title: Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5′UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication

    Journal: Viruses

    doi: 10.3390/v11010017

    Disruption in the 5′UTR nt95–110:NS5B nt8528–8543 duplex reduces intracellular HCV RNA levels. ( A ) Huh-7.5 cells transfected with HCV genomic RNA (WT) or HCV genomic RNA containing mutations at 5′UTR nt 95–110 and NS5B nt 8528–8543 express NS5B antigen 24 h post-transfection. Scale bar = 50 µm. ( B ) Plots represent the average number of HCV RNA genome copies ± SE in 1 μg of cellular RNA at 48 h post-transfection. p -values ≤ 0.05 (*) or ≤ 0.01 (**) were determined by the Student’s t -test and represent four or more independent experiments.
    Figure Legend Snippet: Disruption in the 5′UTR nt95–110:NS5B nt8528–8543 duplex reduces intracellular HCV RNA levels. ( A ) Huh-7.5 cells transfected with HCV genomic RNA (WT) or HCV genomic RNA containing mutations at 5′UTR nt 95–110 and NS5B nt 8528–8543 express NS5B antigen 24 h post-transfection. Scale bar = 50 µm. ( B ) Plots represent the average number of HCV RNA genome copies ± SE in 1 μg of cellular RNA at 48 h post-transfection. p -values ≤ 0.05 (*) or ≤ 0.01 (**) were determined by the Student’s t -test and represent four or more independent experiments.

    Techniques Used: Transfection

    Disruption in the 5′UTR nt 95–110:NS5B nt 8528–8543 duplex reduced progeny virus titers. Huh-7.5 cells were infected with HCV parental virus (WT) or HCV containing mutations at 5′UTR nt 95–110 and NS5B nt 8528–8543. Plots represent the average number of infectious virus titers as focus-forming units (FFU)/mL or HCV RNA genome copies ± SE in 1 μg of cellular RNA taken at 48 h post-infection. p -values ≤ 0.05 (*) or ≤ 0.01 (**) were determined by the Student’s t -test and represent six independent experiments.
    Figure Legend Snippet: Disruption in the 5′UTR nt 95–110:NS5B nt 8528–8543 duplex reduced progeny virus titers. Huh-7.5 cells were infected with HCV parental virus (WT) or HCV containing mutations at 5′UTR nt 95–110 and NS5B nt 8528–8543. Plots represent the average number of infectious virus titers as focus-forming units (FFU)/mL or HCV RNA genome copies ± SE in 1 μg of cellular RNA taken at 48 h post-infection. p -values ≤ 0.05 (*) or ≤ 0.01 (**) were determined by the Student’s t -test and represent six independent experiments.

    Techniques Used: Infection

    2) Product Images from "Complement C1q stimulates the progression of hepatocellular tumor through the activation of discoidin domain receptor 1"

    Article Title: Complement C1q stimulates the progression of hepatocellular tumor through the activation of discoidin domain receptor 1

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23240-6

    Knockdown of DDR1 attenuated the C1q-induced migration and invasion of HepG2 cells. Evaluation of DDR1 protein levels by western blotting after transfection for 36 h with scrambled (siCtrl) or DDR1-specific siRNAs ( A ). Control or DDR1 siRNA-transfected cells were cultured in 48-well plastic plates and allowed to adhere. A linear wound was then created in each well using a pipette and cells were treated with or without C1q to initiate migration ( B ). Transfected cells were cultured in transwell inserts in the presence or absence of C1q for 18 h and invading cells were counted as described in Materials and Methods ( E ). Bar diagrams representing migration distances ( C ), and counts of migrating cells ( D ) and invading cells ( F ). * P
    Figure Legend Snippet: Knockdown of DDR1 attenuated the C1q-induced migration and invasion of HepG2 cells. Evaluation of DDR1 protein levels by western blotting after transfection for 36 h with scrambled (siCtrl) or DDR1-specific siRNAs ( A ). Control or DDR1 siRNA-transfected cells were cultured in 48-well plastic plates and allowed to adhere. A linear wound was then created in each well using a pipette and cells were treated with or without C1q to initiate migration ( B ). Transfected cells were cultured in transwell inserts in the presence or absence of C1q for 18 h and invading cells were counted as described in Materials and Methods ( E ). Bar diagrams representing migration distances ( C ), and counts of migrating cells ( D ) and invading cells ( F ). * P

    Techniques Used: Migration, Western Blot, Transfection, Cell Culture, Transferring

    C1q induced the expressions of MMPs in HepG2 cells. The effects of C1q on the expressions of MMP2 and MMP9 in HepG2 cells were evaluated by real-time PCR. ( A ) Real-time PCR for MMP2 in C1q treated HepG2 cells. Cells were treated with or without the indicated doses of C1q for 6 h, harvested, total RNA was prepared, and reverse transcription reactions were performed using SYBR Select Master Mix Reagent. Real-time PCR was then used to assess MMP2 expressions. ( B ) Real-time PCR for MMP9 in C1q treated HepG2 cells. Cells were transfected with control (siCtrl) or DDR1-specific siRNA for 36 h and then treated with or without C1q for 6 h. The mRNA expressions of MMP2 ( C ) and MMP9 ( D ) were determined by real-time PCR. * P
    Figure Legend Snippet: C1q induced the expressions of MMPs in HepG2 cells. The effects of C1q on the expressions of MMP2 and MMP9 in HepG2 cells were evaluated by real-time PCR. ( A ) Real-time PCR for MMP2 in C1q treated HepG2 cells. Cells were treated with or without the indicated doses of C1q for 6 h, harvested, total RNA was prepared, and reverse transcription reactions were performed using SYBR Select Master Mix Reagent. Real-time PCR was then used to assess MMP2 expressions. ( B ) Real-time PCR for MMP9 in C1q treated HepG2 cells. Cells were transfected with control (siCtrl) or DDR1-specific siRNA for 36 h and then treated with or without C1q for 6 h. The mRNA expressions of MMP2 ( C ) and MMP9 ( D ) were determined by real-time PCR. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Transfection

    3) Product Images from "Trends in bacterial and fungal communities in ant nests observed with Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and Next Generation Sequencing (NGS) techniques—validity and compatibility in ecological studies"

    Article Title: Trends in bacterial and fungal communities in ant nests observed with Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and Next Generation Sequencing (NGS) techniques—validity and compatibility in ecological studies

    Journal: PeerJ

    doi: 10.7717/peerj.5289

    Correlation (Pearson rho ) of T-RFs with the equivalent number of the most abundant OTUs, both ranked highest to lowest. (A) HaeIII, (B) MspI generated bacterial T-RFs, (C) and (D) HaeIII and MspI generated fungal T-RFs respectively.
    Figure Legend Snippet: Correlation (Pearson rho ) of T-RFs with the equivalent number of the most abundant OTUs, both ranked highest to lowest. (A) HaeIII, (B) MspI generated bacterial T-RFs, (C) and (D) HaeIII and MspI generated fungal T-RFs respectively.

    Techniques Used: Generated

    4) Product Images from "Increased neutrophil extracellular traps activate NLRP3 and inflammatory macrophages in adult-onset Still’s disease"

    Article Title: Increased neutrophil extracellular traps activate NLRP3 and inflammatory macrophages in adult-onset Still’s disease

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1800-z

    NET DNA activated macrophages are pivotal to the proinflammatory response in AOSD. a THP-1-derived macrophages were cultured with purified neutrophil extracellular trap (NET) DNA. After 4 h the cells were stained for cell surface markers using specific monoclonal antibodies or the corresponding isotype controls (right panels). Cells were analyzed using flow cytometry. The percentages of proinflammatory macrophages (CD68 + CD86 + ) are indicated (left). The levels of mRNA and protein of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in these THP-1-derived macrophages were measured using RT-PCR ( b ) and ELISA analysis ( c ). The histograms show the means ± SD. * P
    Figure Legend Snippet: NET DNA activated macrophages are pivotal to the proinflammatory response in AOSD. a THP-1-derived macrophages were cultured with purified neutrophil extracellular trap (NET) DNA. After 4 h the cells were stained for cell surface markers using specific monoclonal antibodies or the corresponding isotype controls (right panels). Cells were analyzed using flow cytometry. The percentages of proinflammatory macrophages (CD68 + CD86 + ) are indicated (left). The levels of mRNA and protein of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in these THP-1-derived macrophages were measured using RT-PCR ( b ) and ELISA analysis ( c ). The histograms show the means ± SD. * P

    Techniques Used: Derivative Assay, Cell Culture, Purification, Staining, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Elevated levels of cell-free DNA and NET-DNA complexes in the circulation of patients with active AOSD. a The concentration of cell-free DNA, citrullinated histone 3 (citH3)-DNA, neutrophil elastase (NE)-DNA, and myeloperoxidase (MPO)-DNA complexes in the sera of patients with adult-onset Still’s disease (AOSD; n = 73) or healthy controls (HC; n = 40) were determined using PicoGreen. b Correlation of sera cell-free DNA with citH3-DNA, NE-DNA, and MPO-DNA complexes was analyzed. c Levels of sera cell-free DNA, citH3-DNA, NE-DNA, and MPO-DNA complexes in AOSD patients with active disease and inactive disease were measured using PicoGreen. d Reduced levels of cell-free DNA, citH3-DNA, NE-DNA, and MPO-DNA complexes in 11 AOSD patients after treatment. The results show the means ± SD. * P
    Figure Legend Snippet: Elevated levels of cell-free DNA and NET-DNA complexes in the circulation of patients with active AOSD. a The concentration of cell-free DNA, citrullinated histone 3 (citH3)-DNA, neutrophil elastase (NE)-DNA, and myeloperoxidase (MPO)-DNA complexes in the sera of patients with adult-onset Still’s disease (AOSD; n = 73) or healthy controls (HC; n = 40) were determined using PicoGreen. b Correlation of sera cell-free DNA with citH3-DNA, NE-DNA, and MPO-DNA complexes was analyzed. c Levels of sera cell-free DNA, citH3-DNA, NE-DNA, and MPO-DNA complexes in AOSD patients with active disease and inactive disease were measured using PicoGreen. d Reduced levels of cell-free DNA, citH3-DNA, NE-DNA, and MPO-DNA complexes in 11 AOSD patients after treatment. The results show the means ± SD. * P

    Techniques Used: Concentration Assay

    AOSD NETs are potent triggers of NLRP3 inflammasomes. After stimulation with neutrophil extracellular trap (NET) DNA from adult-onset Still’s disease (AOSD) and healthy controls (HC), mitochondrial DNA (mtDNA), genomic DNA g(DNA), and RNA, the expression ( a ) and secretion ( b ) of interleukin (IL)-1β and IL-18 in THP-1 monocytes were measured using RT-PCR and ELISA, respectively. c Representative immunoblot analysis for NLRP3 inflammasomes in THP-1 monocytes. The images are representative of three independent Western blot experiments. d After stimulation with NET DNA and non-NET source nucleic acids shown above, the expression of IL-1β and IL-18 in PBMC-derived CD14 + monocytes from healthy controls was measured using RT-PCR. e The expression of IL-1β, pro-IL-1β, and NLRP3 in cell lysates (LYS) and IL-1β in supernatants (SN) from THP-1 monocytes after stimulation with NET-DNA in the presence of the NLRP3 inhibitor MCC950 and DNase I was measured by Western blot. The images are representative of three independent Western blot experiments. The histograms show the means ± SD. * P
    Figure Legend Snippet: AOSD NETs are potent triggers of NLRP3 inflammasomes. After stimulation with neutrophil extracellular trap (NET) DNA from adult-onset Still’s disease (AOSD) and healthy controls (HC), mitochondrial DNA (mtDNA), genomic DNA g(DNA), and RNA, the expression ( a ) and secretion ( b ) of interleukin (IL)-1β and IL-18 in THP-1 monocytes were measured using RT-PCR and ELISA, respectively. c Representative immunoblot analysis for NLRP3 inflammasomes in THP-1 monocytes. The images are representative of three independent Western blot experiments. d After stimulation with NET DNA and non-NET source nucleic acids shown above, the expression of IL-1β and IL-18 in PBMC-derived CD14 + monocytes from healthy controls was measured using RT-PCR. e The expression of IL-1β, pro-IL-1β, and NLRP3 in cell lysates (LYS) and IL-1β in supernatants (SN) from THP-1 monocytes after stimulation with NET-DNA in the presence of the NLRP3 inhibitor MCC950 and DNase I was measured by Western blot. The images are representative of three independent Western blot experiments. The histograms show the means ± SD. * P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay

    Enriched mitochondrial DNA in AOSD NETs and plasma. Mitochondrial copy numbers from neutrophil extracellular traps (NETs) ( a ) and plasma ( b ) were determined using RT-PCR. Symbols represent individual samples; horizontal and vertical lines show the means ± SD. * P
    Figure Legend Snippet: Enriched mitochondrial DNA in AOSD NETs and plasma. Mitochondrial copy numbers from neutrophil extracellular traps (NETs) ( a ) and plasma ( b ) were determined using RT-PCR. Symbols represent individual samples; horizontal and vertical lines show the means ± SD. * P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    ROS support enhanced NETosis in AOSD patients [ 39 ]. Cytoplasmic total reactive oxygen species (ROS) stained with DCFH-DA after the activation of neutrophils with 20 nM phorbol myristate acetate (PMA) for 3.5 h was detected using flow plates ( a ) and flow cytometry ( b and c ); the results show the relative fluorescence intensity, ROS-positive neutrophils, and a representative histogram. Mitochondrial ROS production in neutrophils stimulated with 20 nM PMA for 3.5 h and stained with the red fluorescent mitochondrial superoxide indicator, Mito SOX, was determined using flow cytometry ( d ) and immunofluorescence ( e ). Neutrophils from subjects with adult-onset Still’s disease (AOSD; n = 10) were stimulated with 20 nM PMA in the presence of the indicated ROS inhibitor. f , g Sytox green immunofluorescence analysis was performed, and images of neutrophil extracellular trap (NET) formation from each culture were obtained. h The quantification of the DNA concentration was achieved using PicoGreen. The figures are representative of six independent experiments. Scale bars = 20 μm. The histograms show the means ± SD. * P
    Figure Legend Snippet: ROS support enhanced NETosis in AOSD patients [ 39 ]. Cytoplasmic total reactive oxygen species (ROS) stained with DCFH-DA after the activation of neutrophils with 20 nM phorbol myristate acetate (PMA) for 3.5 h was detected using flow plates ( a ) and flow cytometry ( b and c ); the results show the relative fluorescence intensity, ROS-positive neutrophils, and a representative histogram. Mitochondrial ROS production in neutrophils stimulated with 20 nM PMA for 3.5 h and stained with the red fluorescent mitochondrial superoxide indicator, Mito SOX, was determined using flow cytometry ( d ) and immunofluorescence ( e ). Neutrophils from subjects with adult-onset Still’s disease (AOSD; n = 10) were stimulated with 20 nM PMA in the presence of the indicated ROS inhibitor. f , g Sytox green immunofluorescence analysis was performed, and images of neutrophil extracellular trap (NET) formation from each culture were obtained. h The quantification of the DNA concentration was achieved using PicoGreen. The figures are representative of six independent experiments. Scale bars = 20 μm. The histograms show the means ± SD. * P

    Techniques Used: Staining, Activation Assay, Flow Cytometry, Cytometry, Fluorescence, Immunofluorescence, Concentration Assay

    5) Product Images from "Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis"

    Article Title: Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis

    Journal: Mobile DNA

    doi: 10.1186/s13100-018-0138-z

    Nuclear localization of L1 ORF1 protein. a Endogenous ORF1p detected in 2102Ep cells by the α-4H1-ORF1 antibody. ORF1p is mostly cytoplasmic where it concentrates in granules and occasionally at the nuclear membrane. It is faintly seen in the nucleoplasm and concentrates in nucleoli of a subset of cells. b , c Exogenously expressed EGFP-tagged ORF1p strongly concentrates at the nuclear membrane and in perinucleolar foci of 5% or fewer human ( b ) U2OS or ( c ) HEK 293T cells with attendant reduction in size and number of cytoplasmic granules. Cotransfected mCherry-PSP1 marks nuclei and is excluded from nucleoli. d Endogenous ORF1p detected by α-4H1-ORF1 also forms discrete nuclear foci in a minor percentage of 2102Ep cells. Selected foci are enlarged in panels to the right. e Alu RNA, tagged with six MS2 coat protein recognition stem loops and expressed from construct pBS 7SL Alu-MS2 (Ya5), was detected by FISH using a Cy3-tagged DNA probe to the MS2 stem loops. Alu RNA colocalizes with nuclear foci marked by EGFP-tagged ORF1p in HEK 293T cells. f Nuclear foci of MS2 stem loop-tagged full-length SVA RNA detected by the Cy3-MS2 DNA probe do not colocalize with foci marked by ORF1p-EGFP. g RNA having 31 tandem G4C2 repeats detected by FISH using a Cy3-conjugated (C4G2) 4 DNA probe induces intense intranuclear or cytoplasmic RNA aggregates that colocalize with ORF1p-EGFP in a minor percentage of HEK 293T cells (nuclear granules are marked by small arrows and cytoplasmic granules by large arrows). Size bars are 10 μm
    Figure Legend Snippet: Nuclear localization of L1 ORF1 protein. a Endogenous ORF1p detected in 2102Ep cells by the α-4H1-ORF1 antibody. ORF1p is mostly cytoplasmic where it concentrates in granules and occasionally at the nuclear membrane. It is faintly seen in the nucleoplasm and concentrates in nucleoli of a subset of cells. b , c Exogenously expressed EGFP-tagged ORF1p strongly concentrates at the nuclear membrane and in perinucleolar foci of 5% or fewer human ( b ) U2OS or ( c ) HEK 293T cells with attendant reduction in size and number of cytoplasmic granules. Cotransfected mCherry-PSP1 marks nuclei and is excluded from nucleoli. d Endogenous ORF1p detected by α-4H1-ORF1 also forms discrete nuclear foci in a minor percentage of 2102Ep cells. Selected foci are enlarged in panels to the right. e Alu RNA, tagged with six MS2 coat protein recognition stem loops and expressed from construct pBS 7SL Alu-MS2 (Ya5), was detected by FISH using a Cy3-tagged DNA probe to the MS2 stem loops. Alu RNA colocalizes with nuclear foci marked by EGFP-tagged ORF1p in HEK 293T cells. f Nuclear foci of MS2 stem loop-tagged full-length SVA RNA detected by the Cy3-MS2 DNA probe do not colocalize with foci marked by ORF1p-EGFP. g RNA having 31 tandem G4C2 repeats detected by FISH using a Cy3-conjugated (C4G2) 4 DNA probe induces intense intranuclear or cytoplasmic RNA aggregates that colocalize with ORF1p-EGFP in a minor percentage of HEK 293T cells (nuclear granules are marked by small arrows and cytoplasmic granules by large arrows). Size bars are 10 μm

    Techniques Used: Construct, Fluorescence In Situ Hybridization

    6) Product Images from "Mouse REC114 is essential for meiotic DNA double-strand break formation and forms a complex with MEI4"

    Article Title: Mouse REC114 is essential for meiotic DNA double-strand break formation and forms a complex with MEI4

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800259

    Quantification of MEI4 foci and co-immunoprecipitation with REC114 (A) Quantification of MEI4 foci (mean ± SD) in leptotene oocytes from E15 Rec114 +/+ and Rec114 −/− females. The numbers of total foci and foci on chromosome axis (co-localized with SYCP3) were 321 ± 93 and 305 ± 83 in in Rec114 +/+ and 88 ± 50 and 82 ± 47 in Rec114 −/− oocytes; n = 48 and 47). P values were calculated with the Mann–Whitney two-tailed test. (B) Quantification of intensity (arbitrary units) of axis-associated MEI4 foci (mean ± SD) in leptotene spermatocytes and oocytes from Rec114 +/+ and Rec114 −/− mice: 0.0164 ± 0.0068 and 0.0094 ± 0.0015 in Rec114 +/+ and Rec114 −/− spermatocytes (n = 12,001 and 3,775, respectively) and 0.0125 ± 0.0054 and 0.0066 ± 0.0009 in Rec114 +/+ and Rec114 −/− oocytes (n = 14,661 and 3,853, respectively). P values were calculated with the Mann–Whitney two-tailed test. (C) Detection of REC114 after MEI4 immunoprecipitation. Total testis extracts from 14 dpp Rec114 +/+ (WT), Mei4 −/− , Spo11 −/− , and Rec114 −/− mice were immunoprecipitated with an anti-MEI4 antibody. Input extracts and immunoprecipitated fractions were probed with anti-REC114 antibody.
    Figure Legend Snippet: Quantification of MEI4 foci and co-immunoprecipitation with REC114 (A) Quantification of MEI4 foci (mean ± SD) in leptotene oocytes from E15 Rec114 +/+ and Rec114 −/− females. The numbers of total foci and foci on chromosome axis (co-localized with SYCP3) were 321 ± 93 and 305 ± 83 in in Rec114 +/+ and 88 ± 50 and 82 ± 47 in Rec114 −/− oocytes; n = 48 and 47). P values were calculated with the Mann–Whitney two-tailed test. (B) Quantification of intensity (arbitrary units) of axis-associated MEI4 foci (mean ± SD) in leptotene spermatocytes and oocytes from Rec114 +/+ and Rec114 −/− mice: 0.0164 ± 0.0068 and 0.0094 ± 0.0015 in Rec114 +/+ and Rec114 −/− spermatocytes (n = 12,001 and 3,775, respectively) and 0.0125 ± 0.0054 and 0.0066 ± 0.0009 in Rec114 +/+ and Rec114 −/− oocytes (n = 14,661 and 3,853, respectively). P values were calculated with the Mann–Whitney two-tailed test. (C) Detection of REC114 after MEI4 immunoprecipitation. Total testis extracts from 14 dpp Rec114 +/+ (WT), Mei4 −/− , Spo11 −/− , and Rec114 −/− mice were immunoprecipitated with an anti-MEI4 antibody. Input extracts and immunoprecipitated fractions were probed with anti-REC114 antibody.

    Techniques Used: Immunoprecipitation, MANN-WHITNEY, Two Tailed Test, Mouse Assay

    Rec114 −/− mice show defects in DSB formation and homologous synapsis. (Α) Immunostaining of γH2AX and SYCP3 in spermatocytes from 13 dpp Rec114 +/− and Rec114 −/− males, and from E15 (15 d of embryonic development) Rec114 +/+ , Rec114 +/− , and Rec114 −/− oocytes. In Rec114 −/− spermatocytes and oocytes, no pachynema could be observed and spermatocytes or oocytes with partially synapsed chromosomes were defined as zygotene-like. Scale bar, 10 μm. (B) Quantification of the total γH2AX signal per nucleus (mean ± SD; a.u, arbitrary units) on spreads from leptotene spermatocytes (13 dpp) and from leptotene oocytes (E15): 2,597 ± 1,261 and 165 ± 95 in Rec114 +/− and Rec114 −/− males, respectively (n = 53 and 50); 248 ± 187 and 23 ± 7 in Rec114 +/+ and Rec114 −/− females, respectively (n = 48 and 47). P values were calculated with the Mann–Whitney two-tailed test. (C) Immunostaining of DMC1 and SYCP3 in spermatocytes from 15 dpp Rec114 +/+ and Rec114 −/− males. Scale bar, 10 μm. (D) Quantification of DMC1 foci (mean ± SD) in leptotene and zygotene spermatocytes from Rec114 +/+ and Rec114 −/− mice (178.6 ± 39.9 and 17.2 ± 23.0 in Rec114 +/+ and Rec114 −/− males, respectively; n = 71 and 59). P
    Figure Legend Snippet: Rec114 −/− mice show defects in DSB formation and homologous synapsis. (Α) Immunostaining of γH2AX and SYCP3 in spermatocytes from 13 dpp Rec114 +/− and Rec114 −/− males, and from E15 (15 d of embryonic development) Rec114 +/+ , Rec114 +/− , and Rec114 −/− oocytes. In Rec114 −/− spermatocytes and oocytes, no pachynema could be observed and spermatocytes or oocytes with partially synapsed chromosomes were defined as zygotene-like. Scale bar, 10 μm. (B) Quantification of the total γH2AX signal per nucleus (mean ± SD; a.u, arbitrary units) on spreads from leptotene spermatocytes (13 dpp) and from leptotene oocytes (E15): 2,597 ± 1,261 and 165 ± 95 in Rec114 +/− and Rec114 −/− males, respectively (n = 53 and 50); 248 ± 187 and 23 ± 7 in Rec114 +/+ and Rec114 −/− females, respectively (n = 48 and 47). P values were calculated with the Mann–Whitney two-tailed test. (C) Immunostaining of DMC1 and SYCP3 in spermatocytes from 15 dpp Rec114 +/+ and Rec114 −/− males. Scale bar, 10 μm. (D) Quantification of DMC1 foci (mean ± SD) in leptotene and zygotene spermatocytes from Rec114 +/+ and Rec114 −/− mice (178.6 ± 39.9 and 17.2 ± 23.0 in Rec114 +/+ and Rec114 −/− males, respectively; n = 71 and 59). P

    Techniques Used: Mouse Assay, Immunostaining, MANN-WHITNEY, Two Tailed Test

    Reduction of DSB repair foci is not due to absence of SPO11 in Rec114 −/− spermatocytes (A) Immunostaining of RAD51 and SYCP3 in spermatocytes from 15 dpp Rec114 +/+ and Rec114 −/− mice. Scale bar, 10 μm. (B) Detection of SPO11 in Rec114 −/− testis extracts. Immunoprecipitation of SPO11 in testis extracts from WT, Rec114 −/− , Spo11 −/− , and Spo11 YF/YF adult mice. Only the SPO11β variant (44KD) is detected in these conditions.
    Figure Legend Snippet: Reduction of DSB repair foci is not due to absence of SPO11 in Rec114 −/− spermatocytes (A) Immunostaining of RAD51 and SYCP3 in spermatocytes from 15 dpp Rec114 +/+ and Rec114 −/− mice. Scale bar, 10 μm. (B) Detection of SPO11 in Rec114 −/− testis extracts. Immunoprecipitation of SPO11 in testis extracts from WT, Rec114 −/− , Spo11 −/− , and Spo11 YF/YF adult mice. Only the SPO11β variant (44KD) is detected in these conditions.

    Techniques Used: Immunostaining, Mouse Assay, Immunoprecipitation, Variant Assay

    REC114 is required for robust MEI4 foci localization. (A) Immunostaining of IHO1 and SYCP3 in early prophase spermatocytes from 13 dpp Rec114 +/− and Rec114 −/− males. Scale bar, 10 μm. (B) Immunostaining of MEI4 and SYCP3 in early prophase spermatocytes from 13 dpp Rec114 +/− and Rec114 −/− males. Scale bar, 10 μm. (C) Quantification of MEI4 foci in leptotene spermatocytes from 13 dpp Rec114 +/− , Rec114 +/+ , Rec114 −/− , and Mei4 −/− males. The numbers (mean ± SD) of total foci and of foci on chromosome axes (co-localized with SYCP3) were: 248 ± 59 and 226 ± 51 for Rec114 +/− (n = 53 nuclei), 256 ± 78 and 241 ± 68 for Rec114 +/+ (n = 27), 109 ± 37 and 76 ± 29 for Rec114 −/− (n = 50), 45 ± 25, and 23 ± 16 for Mei4 −/− (n = 20), respectively. P values were calculated with the Mann–Whitney two-tailed test.
    Figure Legend Snippet: REC114 is required for robust MEI4 foci localization. (A) Immunostaining of IHO1 and SYCP3 in early prophase spermatocytes from 13 dpp Rec114 +/− and Rec114 −/− males. Scale bar, 10 μm. (B) Immunostaining of MEI4 and SYCP3 in early prophase spermatocytes from 13 dpp Rec114 +/− and Rec114 −/− males. Scale bar, 10 μm. (C) Quantification of MEI4 foci in leptotene spermatocytes from 13 dpp Rec114 +/− , Rec114 +/+ , Rec114 −/− , and Mei4 −/− males. The numbers (mean ± SD) of total foci and of foci on chromosome axes (co-localized with SYCP3) were: 248 ± 59 and 226 ± 51 for Rec114 +/− (n = 53 nuclei), 256 ± 78 and 241 ± 68 for Rec114 +/+ (n = 27), 109 ± 37 and 76 ± 29 for Rec114 −/− (n = 50), 45 ± 25, and 23 ± 16 for Mei4 −/− (n = 20), respectively. P values were calculated with the Mann–Whitney two-tailed test.

    Techniques Used: Immunostaining, MANN-WHITNEY, Two Tailed Test

    (A) RT-PCR analysis on RNA extracted from testes of Rec114 +/+ and Rec114 −/− 14 dpp mice. Controls are PCR without cDNA. The RT-PCR products were separated by agarose gel electrophoresis. Purified PCR products that were sequenced are highlighted in red rectangles. With primer pairs 16U21/644L22 and 16U21/688L24, the PCR products obtained from Rec114 −/− mice migrate faster compared with Rec114 +/+ mice because of the deletion of DNA sequences from exon 3 and 4 and insertion of a 113b DNA sequence from the cassette (EnSA). (B) Western blot analysis of total testis extracts from WT, 13 dpp and Rec114 −/− , 12 dpp after immunoprecipitation with anti-REC114 antibodies, and detection with the same antibody. (C) Testis weight is reduced in Rec114 −/− mice compared with Rec114 +/+ and Rec114 +/− mice. Body and testis weights (mean ± SD) were measured in 8- to 10-wk-old Rec114 +/+ (testis weight 90 ± 5.7; n = 6), Rec114 +/− (testis weight 94.6 ± 3.6 mg; n = 6), and Rec114 −/− (testis weight 25.8 ± 4.3 mg; n = 6) males. P values were calculated with the Mann–Whitney two-tailed test.
    Figure Legend Snippet: (A) RT-PCR analysis on RNA extracted from testes of Rec114 +/+ and Rec114 −/− 14 dpp mice. Controls are PCR without cDNA. The RT-PCR products were separated by agarose gel electrophoresis. Purified PCR products that were sequenced are highlighted in red rectangles. With primer pairs 16U21/644L22 and 16U21/688L24, the PCR products obtained from Rec114 −/− mice migrate faster compared with Rec114 +/+ mice because of the deletion of DNA sequences from exon 3 and 4 and insertion of a 113b DNA sequence from the cassette (EnSA). (B) Western blot analysis of total testis extracts from WT, 13 dpp and Rec114 −/− , 12 dpp after immunoprecipitation with anti-REC114 antibodies, and detection with the same antibody. (C) Testis weight is reduced in Rec114 −/− mice compared with Rec114 +/+ and Rec114 +/− mice. Body and testis weights (mean ± SD) were measured in 8- to 10-wk-old Rec114 +/+ (testis weight 90 ± 5.7; n = 6), Rec114 +/− (testis weight 94.6 ± 3.6 mg; n = 6), and Rec114 −/− (testis weight 25.8 ± 4.3 mg; n = 6) males. P values were calculated with the Mann–Whitney two-tailed test.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Purification, Sequencing, Western Blot, Immunoprecipitation, MANN-WHITNEY, Two Tailed Test

    MEI4, REC114 and IHO1 form a complex. (A) Immunostaining of REC114 and SYCP3 in early prophase spermatocytes from Rec114 +/+ (14 dpp), Mei4 −/− (12 dpp), and Rec114 −/− (14 dpp) males. Scale bar, 10 μm. (B) Quantification of REC114 foci in leptotene spermatocytes from 13 dpp Rec114 +/+ , Mei4 −/− , Spo11 −/− , and Rec114 −/− males. The numbers (mean ± SD) of total foci and of foci on chromosome axis (co-localized with SYCP3) were: 245 ± 58 and 187 ± 47 for Rec114 +/+ (n = 83 nuclei), 241 ± 68 and 189 ± 55 for Spo11 −/− (n = 30), 13 ± 11 and 3 ± 3 for Rec114 −/− (n = 69), and 32 ± 17 and 14 ± 11 for Mei4 −/− (n = 52). P values were calculated with the Mann–Whitney two-tailed test. (C) Co-immunoprecipitation of REC114 and IHO1 with MEI4. Total testis extracts from 14 dpp Rec114 +/+ (WT), Mei4 −/− , Spo11 −/− , and Rec114 −/− mice were immunoprecipitated with an anti-MEI4 antibody. Input extracts were probed with anti-IHO1 and anti-SYCP3 antibodies. MEI4 is not detected in input extracts. Immunoprecipitated fractions were probed with anti-IHO1, anti-MEI4, and anti-REC114 antibodies.
    Figure Legend Snippet: MEI4, REC114 and IHO1 form a complex. (A) Immunostaining of REC114 and SYCP3 in early prophase spermatocytes from Rec114 +/+ (14 dpp), Mei4 −/− (12 dpp), and Rec114 −/− (14 dpp) males. Scale bar, 10 μm. (B) Quantification of REC114 foci in leptotene spermatocytes from 13 dpp Rec114 +/+ , Mei4 −/− , Spo11 −/− , and Rec114 −/− males. The numbers (mean ± SD) of total foci and of foci on chromosome axis (co-localized with SYCP3) were: 245 ± 58 and 187 ± 47 for Rec114 +/+ (n = 83 nuclei), 241 ± 68 and 189 ± 55 for Spo11 −/− (n = 30), 13 ± 11 and 3 ± 3 for Rec114 −/− (n = 69), and 32 ± 17 and 14 ± 11 for Mei4 −/− (n = 52). P values were calculated with the Mann–Whitney two-tailed test. (C) Co-immunoprecipitation of REC114 and IHO1 with MEI4. Total testis extracts from 14 dpp Rec114 +/+ (WT), Mei4 −/− , Spo11 −/− , and Rec114 −/− mice were immunoprecipitated with an anti-MEI4 antibody. Input extracts were probed with anti-IHO1 and anti-SYCP3 antibodies. MEI4 is not detected in input extracts. Immunoprecipitated fractions were probed with anti-IHO1, anti-MEI4, and anti-REC114 antibodies.

    Techniques Used: Immunostaining, MANN-WHITNEY, Two Tailed Test, Immunoprecipitation, Mouse Assay

    Reduction of RPA2 foci in Rec114 −/− meiocytes (A) Immunostaining of replication protein A2 (RPA2) and SYCP3 in 15 dpp spermatocytes and E15 oocytes from Rec114 +/+ and Rec114 −/− mice. Scale bar, 10 μm. (B) Quantification of RPA2 foci (mean ± SD) in leptotene and zygotene oocytes from E15 Rec114 +/− and Rec114 −/− E15 mice (255.9 ± 77.7 and 26.5 ± 14.8, respectively; n = 36 and 35 nuclei). P value was calculated with the Mann–Whitney two-tailed test.
    Figure Legend Snippet: Reduction of RPA2 foci in Rec114 −/− meiocytes (A) Immunostaining of replication protein A2 (RPA2) and SYCP3 in 15 dpp spermatocytes and E15 oocytes from Rec114 +/+ and Rec114 −/− mice. Scale bar, 10 μm. (B) Quantification of RPA2 foci (mean ± SD) in leptotene and zygotene oocytes from E15 Rec114 +/− and Rec114 −/− E15 mice (255.9 ± 77.7 and 26.5 ± 14.8, respectively; n = 36 and 35 nuclei). P value was calculated with the Mann–Whitney two-tailed test.

    Techniques Used: Immunostaining, Mouse Assay, MANN-WHITNEY, Two Tailed Test

    IHO1 localisation does not require MEI4, and MEI4 localisation requires REC114 (A) Immunostaining of IHO1 and SYCP3 in early prophase spermatocytes from 13 dpp Mei4 +/+ and 12 dpp Mei4 −/− males. Scale bar, 10 μm. (B) Immunostaining of MEI4 and SYCP3 in early prophase oocytes from E15 Rec114 +/+ , Rec114 +/− , and Rec114 −/− females. Scale bar, 10 μm.
    Figure Legend Snippet: IHO1 localisation does not require MEI4, and MEI4 localisation requires REC114 (A) Immunostaining of IHO1 and SYCP3 in early prophase spermatocytes from 13 dpp Mei4 +/+ and 12 dpp Mei4 −/− males. Scale bar, 10 μm. (B) Immunostaining of MEI4 and SYCP3 in early prophase oocytes from E15 Rec114 +/+ , Rec114 +/− , and Rec114 −/− females. Scale bar, 10 μm.

    Techniques Used: Immunostaining

    7) Product Images from "Bridging from Intramuscular to Limb Perfusion Delivery of rAAV: Optimization in a Non-human Primate Study"

    Article Title: Bridging from Intramuscular to Limb Perfusion Delivery of rAAV: Optimization in a Non-human Primate Study

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2019.01.013

    Serum AAT c-myc Expression, Muscle Immunohistochemistry for AAT, and RT-PCR (A) Western blot gel image for all time points (please note that several AAV8 day-60 samples were missing). (B) Semi-quantitation of western blot (WB) data represented as mean ± SEM. (C) Immunohistochemistry for AAT at day-60 representative sections for each dosing route for both AAV1 and AAV8. Muscle samples were collected for immunohistochemistry (IHC) from the level of the IM injections in both the quadriceps and gastrocnemius. (D) RT-PCR data from day-60 muscle and liver tissue. The gastrocnemius muscle samples were used for all groups. IM muscle samples were collected from the injection site. IM, intramuscular; IAPD, intra-arterial push and dwell; VLP, venous limb perfusion.
    Figure Legend Snippet: Serum AAT c-myc Expression, Muscle Immunohistochemistry for AAT, and RT-PCR (A) Western blot gel image for all time points (please note that several AAV8 day-60 samples were missing). (B) Semi-quantitation of western blot (WB) data represented as mean ± SEM. (C) Immunohistochemistry for AAT at day-60 representative sections for each dosing route for both AAV1 and AAV8. Muscle samples were collected for immunohistochemistry (IHC) from the level of the IM injections in both the quadriceps and gastrocnemius. (D) RT-PCR data from day-60 muscle and liver tissue. The gastrocnemius muscle samples were used for all groups. IM muscle samples were collected from the injection site. IM, intramuscular; IAPD, intra-arterial push and dwell; VLP, venous limb perfusion.

    Techniques Used: Expressing, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitation Assay, Injection

    8) Product Images from "Smp38 MAP Kinase Regulation in Schistosoma mansoni: Roles in Survival, Oviposition, and Protection Against Oxidative Stress"

    Article Title: Smp38 MAP Kinase Regulation in Schistosoma mansoni: Roles in Survival, Oviposition, and Protection Against Oxidative Stress

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00021

    Smp38 transcript levels in the different life stages and after dsRNAs exposure of Schistosoma mansoni in vitro and mice recovered adult worms. (A) Bar graph depicting the absolute Smp38 transcript levels in the different life stages of S. mansoni . Absolute quantification was used to evaluate the expression levels in cercariae, 2 and 7 days schistosomula, male and female adult worms, and sporocysts are presented as copy number per ng of total RNA. Bars represent standard error of the mean of three technical replicates. (B) Bar graph depicting the relative Smp38 transcript levels in adult worms after 3, 5, 7, and 10 days of electroporation with Smp38.2 dsRNA. Bars represent the values relative to unspecific control (■) or untreated control ( ) (dashed line), for each comparison, data are represented as mean fold-difference (±SE). (C) Bar graph depicting the relative Smp38 transcript levels in schistosomula after 2, 4, and 7 days of exposure to Smp38.1 ( ) or Smp38.2 ( ) dsRNAs. For each dsRNA tested, data are represented as mean fold-difference (±SE) relative to unspecific control (dashed line). (D) Bar graph depicting the relative Smp38 transcript levels in ex-vivo adult worms exposed to Smp38.1 ( ) and Smp38.2 ( ) dsRNAs. Data are represented as mean fold-difference (±SE) relative to untreated control (dashed line). Transcript levels were determined by RT-qPCR and data analyzed using the ΔΔCt method and unpaired t -test with Welch's correction. Asterisks indicates that Smp38 transcript levels exhibit significantly difference relative to the controls; (* p
    Figure Legend Snippet: Smp38 transcript levels in the different life stages and after dsRNAs exposure of Schistosoma mansoni in vitro and mice recovered adult worms. (A) Bar graph depicting the absolute Smp38 transcript levels in the different life stages of S. mansoni . Absolute quantification was used to evaluate the expression levels in cercariae, 2 and 7 days schistosomula, male and female adult worms, and sporocysts are presented as copy number per ng of total RNA. Bars represent standard error of the mean of three technical replicates. (B) Bar graph depicting the relative Smp38 transcript levels in adult worms after 3, 5, 7, and 10 days of electroporation with Smp38.2 dsRNA. Bars represent the values relative to unspecific control (■) or untreated control ( ) (dashed line), for each comparison, data are represented as mean fold-difference (±SE). (C) Bar graph depicting the relative Smp38 transcript levels in schistosomula after 2, 4, and 7 days of exposure to Smp38.1 ( ) or Smp38.2 ( ) dsRNAs. For each dsRNA tested, data are represented as mean fold-difference (±SE) relative to unspecific control (dashed line). (D) Bar graph depicting the relative Smp38 transcript levels in ex-vivo adult worms exposed to Smp38.1 ( ) and Smp38.2 ( ) dsRNAs. Data are represented as mean fold-difference (±SE) relative to untreated control (dashed line). Transcript levels were determined by RT-qPCR and data analyzed using the ΔΔCt method and unpaired t -test with Welch's correction. Asterisks indicates that Smp38 transcript levels exhibit significantly difference relative to the controls; (* p

    Techniques Used: In Vitro, Mouse Assay, Expressing, Electroporation, Ex Vivo, Quantitative RT-PCR

    9) Product Images from "TRPC5 ion channel permeation promotes weight gain in hypercholesterolaemic mice"

    Article Title: TRPC5 ion channel permeation promotes weight gain in hypercholesterolaemic mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37299-8

    Increased adiponectin and reduced inflammatory gene expression in adipose tissue of DNT5 mice. All data were from quantitative real-time PCR analysis of epididymal fat pad RNA after mice had been provided with western-style diet and DOX for 12 weeks. All mice were ApoE −/− . ( A ) Comparison of adiponectin (AdipoQ) and leptin (Lep) mRNA abundance between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 10 Control, N = 14 DNT5). On the right, linear random effects model analysis showing the standardised mean difference and confidence intervals (n/N = 8/10 Control, n/N = 8/14 DNT5). Data above the zero line indicate increase in the DNT5 group. ( B ) Comparison of the inflammatory mediators tumour necrosis factor α (Tnfα), interleukin 6 (Il6), interleukin 1β (Il1β) and C-C motif chemokine ligands 2, 3, 5 and 7 (Ccl2, 3, 5 and 7) mRNA abundance between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 10 Control, N = 14 DNT5). On the right, linear random effects model analysis showing the standardised mean difference and confidence intervals. Data below the zero line indicate decrease in the DNT5 group (n/N = 8/10 Control, n/N = 8/14 DNT5).
    Figure Legend Snippet: Increased adiponectin and reduced inflammatory gene expression in adipose tissue of DNT5 mice. All data were from quantitative real-time PCR analysis of epididymal fat pad RNA after mice had been provided with western-style diet and DOX for 12 weeks. All mice were ApoE −/− . ( A ) Comparison of adiponectin (AdipoQ) and leptin (Lep) mRNA abundance between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 10 Control, N = 14 DNT5). On the right, linear random effects model analysis showing the standardised mean difference and confidence intervals (n/N = 8/10 Control, n/N = 8/14 DNT5). Data above the zero line indicate increase in the DNT5 group. ( B ) Comparison of the inflammatory mediators tumour necrosis factor α (Tnfα), interleukin 6 (Il6), interleukin 1β (Il1β) and C-C motif chemokine ligands 2, 3, 5 and 7 (Ccl2, 3, 5 and 7) mRNA abundance between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 10 Control, N = 14 DNT5). On the right, linear random effects model analysis showing the standardised mean difference and confidence intervals. Data below the zero line indicate decrease in the DNT5 group (n/N = 8/10 Control, n/N = 8/14 DNT5).

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY

    Reduced hepatic gene expression for lipogenesis mediators in DNT5 mice. All data were from mice which had been provided with western-style diet and DOX for 12 weeks. All mice were ApoE −/− . ( A ) Example liver sections stained with H E. Scale bar 100 μm. ( B ) Comparison of lipid content (steatosis) between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 11 Control, N = 14 DNT5). On the right, linear random effects model showing the standardised mean difference and confidence intervals. Data below the zero line indicate decrease in the DNT5 group (n/N = 8/11 Control, n/N = 8/13 DNT5). ( C ) All data were from quantitative real-time PCR analysis of liver RNA. Comparison of expression of sterol regulatory element-binding protein 1c gene (Srebp1c), acetyl-CoA carboxylase 1 gene (Acaca), fatty acid synthase gene (Fasn) and stearoyl-CoA desaturase 1 gene (Scd1) mRNA abundance between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 11 Control, N = 14 DNT5). On the right linear random effects model showing the standardised mean difference and confidence intervals. Data below the zero line indicate decrease in the DNT5 group (n/N = 8/11 Control, n/N = 8/13 DNT5). ( D ) Comparison of non-fasting plasma concentrations of cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides between Controls and DNT5 after 12 weeks of western-style diet and DOX administration. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 11 Control, N = 14 DNT5). On the right, linear random effects model showing the standardised mean difference and confidence intervals (n/N = 8/11 Control, n/N = 8/14 DNT5).
    Figure Legend Snippet: Reduced hepatic gene expression for lipogenesis mediators in DNT5 mice. All data were from mice which had been provided with western-style diet and DOX for 12 weeks. All mice were ApoE −/− . ( A ) Example liver sections stained with H E. Scale bar 100 μm. ( B ) Comparison of lipid content (steatosis) between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 11 Control, N = 14 DNT5). On the right, linear random effects model showing the standardised mean difference and confidence intervals. Data below the zero line indicate decrease in the DNT5 group (n/N = 8/11 Control, n/N = 8/13 DNT5). ( C ) All data were from quantitative real-time PCR analysis of liver RNA. Comparison of expression of sterol regulatory element-binding protein 1c gene (Srebp1c), acetyl-CoA carboxylase 1 gene (Acaca), fatty acid synthase gene (Fasn) and stearoyl-CoA desaturase 1 gene (Scd1) mRNA abundance between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 11 Control, N = 14 DNT5). On the right linear random effects model showing the standardised mean difference and confidence intervals. Data below the zero line indicate decrease in the DNT5 group (n/N = 8/11 Control, n/N = 8/13 DNT5). ( D ) Comparison of non-fasting plasma concentrations of cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides between Controls and DNT5 after 12 weeks of western-style diet and DOX administration. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 11 Control, N = 14 DNT5). On the right, linear random effects model showing the standardised mean difference and confidence intervals (n/N = 8/11 Control, n/N = 8/14 DNT5).

    Techniques Used: Expressing, Mouse Assay, Western Blot, Staining, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Binding Assay

    10) Product Images from "Formaldehyde Induces Mesenteric Artery Relaxation via a Sensitive Transient Receptor Potential Ankyrin-1 (TRPA1) and Endothelium-Dependent Mechanism: Potential Role in Postprandial Hyperemia"

    Article Title: Formaldehyde Induces Mesenteric Artery Relaxation via a Sensitive Transient Receptor Potential Ankyrin-1 (TRPA1) and Endothelium-Dependent Mechanism: Potential Role in Postprandial Hyperemia

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2019.00277

    Role of the TRPA1 channel in formaldehyde (FA)-induced relaxation in phenylephrine (PE) pre-contracted SMA. (A) Representative traces of FA-stimulated relaxation of PE pre-contracted SMA in the absence (Control) and presence of the TRPA1 antagonist, A967079 (3 μM), which was added after PE-induced contraction plateaued and prior to cumulative addition of FA in wild type (WT) SMA. (B) Summary data of FA-induced relaxation in isolated SMA pre-contracted with PE in the absence and presence of TRPA1 antagonist. Immunofluorescence localization of TRPA1 in SMA (C) and in dorsal root ganglion (DRG; D ). Formalin-fixed and paraffin-embedded sections of murine organs were stained with H E (Ci, Di) ; TRPA1 only (green; Cii ) or DAPI only (blue, nuclear stain; Dii ); TRPA1 antibody (green), isolectin (red, endothelium) and DAPI (Ciii) or TRPA1 antibody (red) and DAPI (Diii) ; and, TRPA1 antibody, TRPA1 blocking peptide, isolectin and DAPI (Civ) or TRPA1 antibody, TRPA1 blocking peptide, and DAPI (Div). L, lumen of SMA. All images were at 200× magnification (scale bar = 100 μm). Ei ) Schematic depicting mouse mTrpa1 gene exons (E) 23 to 25 (with introns, I) and their length (in base pairs, bp). TRPA1 mRNA with the spliced exons 23 to 25 makes a 309 bp product as indicated by TRPA1 specific primers used. (Eii) Agarose gel electrophoresis of PCR amplified product from 309 bp cDNA by qRT-PCR (101 bp product from SMA or DRG of WT mouse using TaqMan Assay for TRPA1; ct values indicated). (Eiii) DNA sequence of 309 bp PCR product identified as TRPA1. Sequence of DRG PCR product (309 bp) was used as validation (data not shown). Values are means ± SE of 3–4 preparations. ∗ , P
    Figure Legend Snippet: Role of the TRPA1 channel in formaldehyde (FA)-induced relaxation in phenylephrine (PE) pre-contracted SMA. (A) Representative traces of FA-stimulated relaxation of PE pre-contracted SMA in the absence (Control) and presence of the TRPA1 antagonist, A967079 (3 μM), which was added after PE-induced contraction plateaued and prior to cumulative addition of FA in wild type (WT) SMA. (B) Summary data of FA-induced relaxation in isolated SMA pre-contracted with PE in the absence and presence of TRPA1 antagonist. Immunofluorescence localization of TRPA1 in SMA (C) and in dorsal root ganglion (DRG; D ). Formalin-fixed and paraffin-embedded sections of murine organs were stained with H E (Ci, Di) ; TRPA1 only (green; Cii ) or DAPI only (blue, nuclear stain; Dii ); TRPA1 antibody (green), isolectin (red, endothelium) and DAPI (Ciii) or TRPA1 antibody (red) and DAPI (Diii) ; and, TRPA1 antibody, TRPA1 blocking peptide, isolectin and DAPI (Civ) or TRPA1 antibody, TRPA1 blocking peptide, and DAPI (Div). L, lumen of SMA. All images were at 200× magnification (scale bar = 100 μm). Ei ) Schematic depicting mouse mTrpa1 gene exons (E) 23 to 25 (with introns, I) and their length (in base pairs, bp). TRPA1 mRNA with the spliced exons 23 to 25 makes a 309 bp product as indicated by TRPA1 specific primers used. (Eii) Agarose gel electrophoresis of PCR amplified product from 309 bp cDNA by qRT-PCR (101 bp product from SMA or DRG of WT mouse using TaqMan Assay for TRPA1; ct values indicated). (Eiii) DNA sequence of 309 bp PCR product identified as TRPA1. Sequence of DRG PCR product (309 bp) was used as validation (data not shown). Values are means ± SE of 3–4 preparations. ∗ , P

    Techniques Used: Isolation, Immunofluorescence, Staining, Blocking Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, TaqMan Assay, Sequencing

    11) Product Images from "Transactivation of a Viral Target Gene by Herpes Simplex Virus ICP27 Is Posttranscriptional and Does Not Require the Endogenous Promoter or Polyadenylation Site"

    Article Title: Transactivation of a Viral Target Gene by Herpes Simplex Virus ICP27 Is Posttranscriptional and Does Not Require the Endogenous Promoter or Polyadenylation Site

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.18.9872-9884.2003

    ICP27 transactivates the gC gene, in combination with ICP4 and ICP0. (A). Induction of gC protein expression. Vero cells were transfected with pUC19 only (lane 3) or with pgC (lanes 4 to 7). Transfections also included pSG1, which encodes ICP4 and ICP0 (lane 5); pC27, which encodes ICP27 (lane 6); or pSG1 plus pC27 (lane 7). After 2 days, cell lysates were prepared and equal amounts were subjected to immunoblotting using a monoclonal antibody specific for gC. Lane 1 shows protein size standards; lane 2 contains protein extract from HSV-1-infected Vero cells, harvested at 6 hpi. (B) Induction of gC mRNA expression. Vero cells were transfected with the plasmids indicated, as in panel A. RNA was prepared at 2 days posttransfection, and equal amounts were subjected to Northern analysis using a gC gene-specific probe. Lane 1 contains RNA from HSV-1-infected Vero cells. (C) ICP8 gene expression is not affected by ICP27. Vero cells were transfected as in panel A, except that plasmid pSG18, which encodes ICP8, replaced pgC. Protein analysis was carried out as in panel A, except the blot was probed with an ICP8-specific monoclonal antibody.
    Figure Legend Snippet: ICP27 transactivates the gC gene, in combination with ICP4 and ICP0. (A). Induction of gC protein expression. Vero cells were transfected with pUC19 only (lane 3) or with pgC (lanes 4 to 7). Transfections also included pSG1, which encodes ICP4 and ICP0 (lane 5); pC27, which encodes ICP27 (lane 6); or pSG1 plus pC27 (lane 7). After 2 days, cell lysates were prepared and equal amounts were subjected to immunoblotting using a monoclonal antibody specific for gC. Lane 1 shows protein size standards; lane 2 contains protein extract from HSV-1-infected Vero cells, harvested at 6 hpi. (B) Induction of gC mRNA expression. Vero cells were transfected with the plasmids indicated, as in panel A. RNA was prepared at 2 days posttransfection, and equal amounts were subjected to Northern analysis using a gC gene-specific probe. Lane 1 contains RNA from HSV-1-infected Vero cells. (C) ICP8 gene expression is not affected by ICP27. Vero cells were transfected as in panel A, except that plasmid pSG18, which encodes ICP8, replaced pgC. Protein analysis was carried out as in panel A, except the blot was probed with an ICP8-specific monoclonal antibody.

    Techniques Used: Expressing, Transfection, Pyrolysis Gas Chromatography, Infection, Northern Blot, Plasmid Preparation

    12) Product Images from "Human Cytomegalovirus-Encoded Interleukin-10 Homolog Inhibits Maturation of Dendritic Cells and Alters Their Functionality"

    Article Title: Human Cytomegalovirus-Encoded Interleukin-10 Homolog Inhibits Maturation of Dendritic Cells and Alters Their Functionality

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.16.8720-8731.2004

    Expression kinetics of HCMV ORF UL111A during viral replication. (A) UL111A is transcribed with late gene kinetics. MRC-5 cells were infected in triplicate with HCMV AD169 at an MOI of 1 and cultured in the presence (open symbols) or absence (filled symbols) of foscarnet (PFA; 167 μM) and ganciclovir (GCV; 4 μM). Supernatants were collected daily to quantify progeny virus titers by standard plaque assays (shown as lines), and total RNA was extracted for cDNA synthesis and quantitative real-time PCR analyses. The relative copy numbers of UL111A cDNA after normalization with GAPDH copy numbers are shown as bars. (B) Accumulation of cmvIL-10 protein in the supernatants of infected cultures. Cells were infected with HCMV strain AD169 (filled symbols) or Toledo (open symbols) at an MOI of 0.1. Supernatants were collected at indicated time points to measure virus titers (shown as lines) and cmvIL-10 protein concentrations (shown as bars). Shown are mean values ± standard deviations for triplicate cultures.
    Figure Legend Snippet: Expression kinetics of HCMV ORF UL111A during viral replication. (A) UL111A is transcribed with late gene kinetics. MRC-5 cells were infected in triplicate with HCMV AD169 at an MOI of 1 and cultured in the presence (open symbols) or absence (filled symbols) of foscarnet (PFA; 167 μM) and ganciclovir (GCV; 4 μM). Supernatants were collected daily to quantify progeny virus titers by standard plaque assays (shown as lines), and total RNA was extracted for cDNA synthesis and quantitative real-time PCR analyses. The relative copy numbers of UL111A cDNA after normalization with GAPDH copy numbers are shown as bars. (B) Accumulation of cmvIL-10 protein in the supernatants of infected cultures. Cells were infected with HCMV strain AD169 (filled symbols) or Toledo (open symbols) at an MOI of 0.1. Supernatants were collected at indicated time points to measure virus titers (shown as lines) and cmvIL-10 protein concentrations (shown as bars). Shown are mean values ± standard deviations for triplicate cultures.

    Techniques Used: Expressing, Infection, Cell Culture, Real-time Polymerase Chain Reaction

    13) Product Images from "Quantitative Analysis of Retromer Complex-Related Genes during Embryo Development in the Mouse"

    Article Title: Quantitative Analysis of Retromer Complex-Related Genes during Embryo Development in the Mouse

    Journal: Molecules and Cells

    doi: 10.1007/s10059-011-0272-7

    Expression of retromer complex-related genes in mouse embryonic fibroblast (MEF) and Vps26a -/- MEF cells. (A) Vps26a transcript was not detected in Vps26a -/- MEF cells. (B) Relative expression of retromer complexrelated genes. Each value represents the mean ± SD. 1, MEF; 2, Vps26a -/- ; 3, MEF.
    Figure Legend Snippet: Expression of retromer complex-related genes in mouse embryonic fibroblast (MEF) and Vps26a -/- MEF cells. (A) Vps26a transcript was not detected in Vps26a -/- MEF cells. (B) Relative expression of retromer complexrelated genes. Each value represents the mean ± SD. 1, MEF; 2, Vps26a -/- ; 3, MEF.

    Techniques Used: Expressing

    14) Product Images from "Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids"

    Article Title: Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

    Journal: Journal of Virology

    doi: 10.1128/JVI.00582-13

    Identification of the molecular target of sulfamoylbenzamide derivatives by genetic complementation assays between HBV and WHV. HepG2 cells were cotransfected with plasmids pCMVHBV/C − and pcDNA3/HBVcore (A), pCMVHBV/C − and pcDNA3/WHVcore (B), or pCMVHBV/C − and pCMVWHV/P − (C and D). Six hours after transfection, the cells were mock treated or treated with 10 μM lamivudine, 2.5 μM Bay 41-4109, 25 μM AT-61, or 10 μM DVR-01 for 3 days. Cytoplasmic core-associated viral DNA replication intermediates were extracted and determined by Southern blot hybridizations with a 32 P-labeled full-length riboprobe specific to the minus strand of HBV (A, B, and C) and WHV (D). RC and SS indicate relaxed circular and single-stranded HBV DNA, respectively. The sources of the capsid protein, polymerase, pgRNA, and probe for hybridization for each of the experiments are presented below each blot.
    Figure Legend Snippet: Identification of the molecular target of sulfamoylbenzamide derivatives by genetic complementation assays between HBV and WHV. HepG2 cells were cotransfected with plasmids pCMVHBV/C − and pcDNA3/HBVcore (A), pCMVHBV/C − and pcDNA3/WHVcore (B), or pCMVHBV/C − and pCMVWHV/P − (C and D). Six hours after transfection, the cells were mock treated or treated with 10 μM lamivudine, 2.5 μM Bay 41-4109, 25 μM AT-61, or 10 μM DVR-01 for 3 days. Cytoplasmic core-associated viral DNA replication intermediates were extracted and determined by Southern blot hybridizations with a 32 P-labeled full-length riboprobe specific to the minus strand of HBV (A, B, and C) and WHV (D). RC and SS indicate relaxed circular and single-stranded HBV DNA, respectively. The sources of the capsid protein, polymerase, pgRNA, and probe for hybridization for each of the experiments are presented below each blot.

    Techniques Used: Transfection, Southern Blot, Labeling, Hybridization

    15) Product Images from "Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids"

    Article Title: Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

    Journal: Journal of Virology

    doi: 10.1128/JVI.00582-13

    Identification of the molecular target of sulfamoylbenzamide derivatives by genetic complementation assays between HBV and WHV. HepG2 cells were cotransfected with plasmids pCMVHBV/C − and pcDNA3/HBVcore (A), pCMVHBV/C − and pcDNA3/WHVcore (B), or pCMVHBV/C − and pCMVWHV/P − (C and D). Six hours after transfection, the cells were mock treated or treated with 10 μM lamivudine, 2.5 μM Bay 41-4109, 25 μM AT-61, or 10 μM DVR-01 for 3 days. Cytoplasmic core-associated viral DNA replication intermediates were extracted and determined by Southern blot hybridizations with a 32 P-labeled full-length riboprobe specific to the minus strand of HBV (A, B, and C) and WHV (D). RC and SS indicate relaxed circular and single-stranded HBV DNA, respectively. The sources of the capsid protein, polymerase, pgRNA, and probe for hybridization for each of the experiments are presented below each blot.
    Figure Legend Snippet: Identification of the molecular target of sulfamoylbenzamide derivatives by genetic complementation assays between HBV and WHV. HepG2 cells were cotransfected with plasmids pCMVHBV/C − and pcDNA3/HBVcore (A), pCMVHBV/C − and pcDNA3/WHVcore (B), or pCMVHBV/C − and pCMVWHV/P − (C and D). Six hours after transfection, the cells were mock treated or treated with 10 μM lamivudine, 2.5 μM Bay 41-4109, 25 μM AT-61, or 10 μM DVR-01 for 3 days. Cytoplasmic core-associated viral DNA replication intermediates were extracted and determined by Southern blot hybridizations with a 32 P-labeled full-length riboprobe specific to the minus strand of HBV (A, B, and C) and WHV (D). RC and SS indicate relaxed circular and single-stranded HBV DNA, respectively. The sources of the capsid protein, polymerase, pgRNA, and probe for hybridization for each of the experiments are presented below each blot.

    Techniques Used: Transfection, Southern Blot, Labeling, Hybridization

    16) Product Images from "NKp30 isoforms and NKp46 transcripts in metastatic melanoma patients: Unique NKp30 pattern in rare melanoma patients with favorable evolution"

    Article Title: NKp30 isoforms and NKp46 transcripts in metastatic melanoma patients: Unique NKp30 pattern in rare melanoma patients with favorable evolution

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2016.1154251

    NCR3 genetic variants in Long Survivors and Progressors Melanoma patients. (A–B) Frequencies of NCR3 genetic variants rs986475 (A) and rs15575842 (B) in HV and progressor or LS melanoma patients. (C–F) Analysis of membrane expression of NKp30 according to the presence of the two NCR3 SNP, rs986475 (C) and rs11575842 (E) in progressor (n = 37, black dots) and 13 LS (n = 13, white dots) melanoma patients. Transcript levels (left panels) or ratios (right panels) of NKp30 isoforms according to rs986475 (D) and rs11575842 (F) SNPs in progressor (n = 104) or LS (n = 13) melanoma patients. Non-parametric Mann–Whitney test was used to compare groups.
    Figure Legend Snippet: NCR3 genetic variants in Long Survivors and Progressors Melanoma patients. (A–B) Frequencies of NCR3 genetic variants rs986475 (A) and rs15575842 (B) in HV and progressor or LS melanoma patients. (C–F) Analysis of membrane expression of NKp30 according to the presence of the two NCR3 SNP, rs986475 (C) and rs11575842 (E) in progressor (n = 37, black dots) and 13 LS (n = 13, white dots) melanoma patients. Transcript levels (left panels) or ratios (right panels) of NKp30 isoforms according to rs986475 (D) and rs11575842 (F) SNPs in progressor (n = 104) or LS (n = 13) melanoma patients. Non-parametric Mann–Whitney test was used to compare groups.

    Techniques Used: Expressing, MANN-WHITNEY

    17) Product Images from "A sensitive real-time PCR based assay to estimate the impact of amino acid substitutions on the competitive replication fitness of human immunodeficiency virus type 1 in cell culture"

    Article Title: A sensitive real-time PCR based assay to estimate the impact of amino acid substitutions on the competitive replication fitness of human immunodeficiency virus type 1 in cell culture

    Journal: Journal of virological methods

    doi: 10.1016/j.jviromet.2012.10.016

    Detection of pNL4-3VifA and pNL4-3VifB in mixtures using common sense primer and probe and vifA- and vifB- specific antisense primers A) Detection of pNL4-3VifA (30 to 3×10 6 copies) using the vifA -specific primer in the presence of 3×10 6 copies of pNL4-3VifB. B) Detection of pNL4-3VifB (30 to 3×10 6 copies) using the vifB -specific primer in the presence of 3×10 6 copies pNL4-3VifA. C) Comparison of the threshold cycles in real-time PCR reactions containing differing amounts of pNL4-3VifA with or without 3×10 6 copies of pNL4-3VifB, using vifA -specific primer. For A) to C), results from triplicate reactions are shown. D) Quantification of serially diluted chimeric virus gagVifA (dilution factors ranged from 1 to 10 6 ) with or without undiluted gagVifB. Serially diluted transfection supernatants of gagVifA were mixed with undiluted transfection supernatants of gagVifB at a 1:1 ratio based on volume. RNA was purified from 50μl gagVifA only samples or gagVifA/gagVifB mixtures. After DNase treatment, cDNA was synthesized using 7μl DNA-free RNA and the PCR-amplifiable copy numbers of gagVifA in 1μ cDNA were determined in real-time PCR using the vifA -specific primer. In addition, the PCR-amplifiable copy number of gagVifB in the mixture was estimated from 1μ cDNA derived from 50μl of the gagVifB only sample using the vifB -specific primer. The copy numbers in the gagVifB and gagVifA only samples in the figure were adjusted (divided by 2) since there were only 25μl of gagVifA and gagVifB each in gagVifA/gagVifB mixtures. Results from duplicate reactions are shown.
    Figure Legend Snippet: Detection of pNL4-3VifA and pNL4-3VifB in mixtures using common sense primer and probe and vifA- and vifB- specific antisense primers A) Detection of pNL4-3VifA (30 to 3×10 6 copies) using the vifA -specific primer in the presence of 3×10 6 copies of pNL4-3VifB. B) Detection of pNL4-3VifB (30 to 3×10 6 copies) using the vifB -specific primer in the presence of 3×10 6 copies pNL4-3VifA. C) Comparison of the threshold cycles in real-time PCR reactions containing differing amounts of pNL4-3VifA with or without 3×10 6 copies of pNL4-3VifB, using vifA -specific primer. For A) to C), results from triplicate reactions are shown. D) Quantification of serially diluted chimeric virus gagVifA (dilution factors ranged from 1 to 10 6 ) with or without undiluted gagVifB. Serially diluted transfection supernatants of gagVifA were mixed with undiluted transfection supernatants of gagVifB at a 1:1 ratio based on volume. RNA was purified from 50μl gagVifA only samples or gagVifA/gagVifB mixtures. After DNase treatment, cDNA was synthesized using 7μl DNA-free RNA and the PCR-amplifiable copy numbers of gagVifA in 1μ cDNA were determined in real-time PCR using the vifA -specific primer. In addition, the PCR-amplifiable copy number of gagVifB in the mixture was estimated from 1μ cDNA derived from 50μl of the gagVifB only sample using the vifB -specific primer. The copy numbers in the gagVifB and gagVifA only samples in the figure were adjusted (divided by 2) since there were only 25μl of gagVifA and gagVifB each in gagVifA/gagVifB mixtures. Results from duplicate reactions are shown.

    Techniques Used: Real-time Polymerase Chain Reaction, Transfection, Purification, Synthesized, Polymerase Chain Reaction, Derivative Assay

    Growth kinetics of gag and env chimeric viruses in dual and mono infections Growth kinetics of gag chimeric viruses in dual (A–C) and mono-infections (D). Growth kinetics of env chimeric viruses in dual (E–K) and mono-infections (L). All dual infections were done in triplicate. In parallel, mono-infection of each competing virus was performed in a single replicate. Supernatants from infections were sampled daily, viral RNA were extracted and cDNA synthesized using 1μl DNase-treated RNA. cDNA copy numbers are reported using real time PCR in duplicate reactions along with standard deviations. The gray areas represent the data used for fitness calculations. Different symbols refer to different culture replicates. Different colored lines refer to different chimeric viruses.
    Figure Legend Snippet: Growth kinetics of gag and env chimeric viruses in dual and mono infections Growth kinetics of gag chimeric viruses in dual (A–C) and mono-infections (D). Growth kinetics of env chimeric viruses in dual (E–K) and mono-infections (L). All dual infections were done in triplicate. In parallel, mono-infection of each competing virus was performed in a single replicate. Supernatants from infections were sampled daily, viral RNA were extracted and cDNA synthesized using 1μl DNase-treated RNA. cDNA copy numbers are reported using real time PCR in duplicate reactions along with standard deviations. The gray areas represent the data used for fitness calculations. Different symbols refer to different culture replicates. Different colored lines refer to different chimeric viruses.

    Techniques Used: Infection, Synthesized, Real-time Polymerase Chain Reaction

    Characterictics of gag and env chimeric founder and mutant viruses A) Mutants and location of mutations in proteins from HIV-1 HXB2 . B) gag mutants. C) env mutants. Viral RNA (~70μl) was purified from 200μl transfection supernatants. After DNase treatment, cDNA was synthesized using 1μl DNA-free RNA and the PCR-amplifiable copy numbers were determined in real-time PCR using specific gag ). TCID 50 ) using PBMC and p24 antigen capture assays.
    Figure Legend Snippet: Characterictics of gag and env chimeric founder and mutant viruses A) Mutants and location of mutations in proteins from HIV-1 HXB2 . B) gag mutants. C) env mutants. Viral RNA (~70μl) was purified from 200μl transfection supernatants. After DNase treatment, cDNA was synthesized using 1μl DNA-free RNA and the PCR-amplifiable copy numbers were determined in real-time PCR using specific gag ). TCID 50 ) using PBMC and p24 antigen capture assays.

    Techniques Used: Mutagenesis, Purification, Transfection, Synthesized, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Detection of pNL4-3VifA and pNL4-3VifB in mixtures using common sense primer and probe and vifA- and vifB- specific antisense primers A) Detection of pNL4-3VifA (30 to 3×10 6 copies) using the vifA -specific primer in the presence of 3×10 6 copies of pNL4-3VifB. B) Detection of pNL4-3VifB (30 to 3×10 6 copies) using the vifB -specific primer in the presence of 3×10 6 copies pNL4-3VifA. C) Comparison of the threshold cycles in real-time PCR reactions containing differing amounts of pNL4-3VifA with or without 3×10 6 copies of pNL4-3VifB, using vifA -specific primer. For A) to C), results from triplicate reactions are shown. D) Quantification of serially diluted chimeric virus gagVifA (dilution factors ranged from 1 to 10 6 ) with or without undiluted gagVifB. Serially diluted transfection supernatants of gagVifA were mixed with undiluted transfection supernatants of gagVifB at a 1:1 ratio based on volume. RNA was purified from 50μl gagVifA only samples or gagVifA/gagVifB mixtures. After DNase treatment, cDNA was synthesized using 7μl DNA-free RNA and the PCR-amplifiable copy numbers of gagVifA in 1μ cDNA were determined in real-time PCR using the vifA -specific primer. In addition, the PCR-amplifiable copy number of gagVifB in the mixture was estimated from 1μ cDNA derived from 50μl of the gagVifB only sample using the vifB -specific primer. The copy numbers in the gagVifB and gagVifA only samples in the figure were adjusted (divided by 2) since there were only 25μl of gagVifA and gagVifB each in gagVifA/gagVifB mixtures. Results from duplicate reactions are shown.
    Figure Legend Snippet: Detection of pNL4-3VifA and pNL4-3VifB in mixtures using common sense primer and probe and vifA- and vifB- specific antisense primers A) Detection of pNL4-3VifA (30 to 3×10 6 copies) using the vifA -specific primer in the presence of 3×10 6 copies of pNL4-3VifB. B) Detection of pNL4-3VifB (30 to 3×10 6 copies) using the vifB -specific primer in the presence of 3×10 6 copies pNL4-3VifA. C) Comparison of the threshold cycles in real-time PCR reactions containing differing amounts of pNL4-3VifA with or without 3×10 6 copies of pNL4-3VifB, using vifA -specific primer. For A) to C), results from triplicate reactions are shown. D) Quantification of serially diluted chimeric virus gagVifA (dilution factors ranged from 1 to 10 6 ) with or without undiluted gagVifB. Serially diluted transfection supernatants of gagVifA were mixed with undiluted transfection supernatants of gagVifB at a 1:1 ratio based on volume. RNA was purified from 50μl gagVifA only samples or gagVifA/gagVifB mixtures. After DNase treatment, cDNA was synthesized using 7μl DNA-free RNA and the PCR-amplifiable copy numbers of gagVifA in 1μ cDNA were determined in real-time PCR using the vifA -specific primer. In addition, the PCR-amplifiable copy number of gagVifB in the mixture was estimated from 1μ cDNA derived from 50μl of the gagVifB only sample using the vifB -specific primer. The copy numbers in the gagVifB and gagVifA only samples in the figure were adjusted (divided by 2) since there were only 25μl of gagVifA and gagVifB each in gagVifA/gagVifB mixtures. Results from duplicate reactions are shown.

    Techniques Used: Real-time Polymerase Chain Reaction, Transfection, Purification, Synthesized, Polymerase Chain Reaction, Derivative Assay

    Characterictics of gag and env chimeric founder and mutant viruses A) Mutants and location of mutations in proteins from HIV-1 HXB2 . B) gag mutants. C) env mutants. Viral RNA (~70μl) was purified from 200μl transfection supernatants. After DNase treatment, cDNA was synthesized using 1μl DNA-free RNA and the PCR-amplifiable copy numbers were determined in real-time PCR using specific gag ). TCID 50 ) using PBMC and p24 antigen capture assays.
    Figure Legend Snippet: Characterictics of gag and env chimeric founder and mutant viruses A) Mutants and location of mutations in proteins from HIV-1 HXB2 . B) gag mutants. C) env mutants. Viral RNA (~70μl) was purified from 200μl transfection supernatants. After DNase treatment, cDNA was synthesized using 1μl DNA-free RNA and the PCR-amplifiable copy numbers were determined in real-time PCR using specific gag ). TCID 50 ) using PBMC and p24 antigen capture assays.

    Techniques Used: Mutagenesis, Purification, Transfection, Synthesized, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    18) Product Images from "EspI regulates the ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis"

    Article Title: EspI regulates the ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis

    Journal: Molecular microbiology

    doi: 10.1111/mmi.12718

    Complementation of the espI ::Tn mutant (A) RT-PCR analysis of espI and eccD 1 cotranscription. Upper panel shows genetic arrangement of espI and eccD 1 , and primer annealing sites. Blue arrows represent the internal primers for espI , green arrows the internal primers for eccD 1 and red arrows the primers spanning the two genes. Lower panel shows RT-PCR analysis of total RNA from wild-type M. tuberculosis Erdman. 2F9 cosmid was used as a positive PCR control. - RT, no reverse transcriptase. (B) Quantitative RT-PCR analysis of mRNA levels of esxA and eccD 1. Relative expression levels were calculated using the ΔΔCt method, normalizing transcript levels to sigA signals. Data are shown as percentage relative to wild-type M. tuberculosis. (C) Immunoblots of cell lysates (CL) at 5 μg/well and culture filtrates (CF) at 10 μg/well of wild-type M. tuberculosis Erdman (WT) and espI ::Tn transformed with indicated plasmids grown in Sauton’s medium without Tween-80 for 5 days. Antibodies used are indicated. (D) Cytotoxicity assay in THP-1 cells infected with wild-type M. tuberculosis Erdman (Erd WT) (black bar) and espI ::Tn (grey bars) transformed with indicated plasmids at MOI of 5. Data represent the means and standard deviation of at least 4 independent experiments. The y-axis indicates cytotoxicity values relative to the uninfected control. Significance in difference compared to wild-type M. tuberculosis was calculated using Student’s t -test. *, p
    Figure Legend Snippet: Complementation of the espI ::Tn mutant (A) RT-PCR analysis of espI and eccD 1 cotranscription. Upper panel shows genetic arrangement of espI and eccD 1 , and primer annealing sites. Blue arrows represent the internal primers for espI , green arrows the internal primers for eccD 1 and red arrows the primers spanning the two genes. Lower panel shows RT-PCR analysis of total RNA from wild-type M. tuberculosis Erdman. 2F9 cosmid was used as a positive PCR control. - RT, no reverse transcriptase. (B) Quantitative RT-PCR analysis of mRNA levels of esxA and eccD 1. Relative expression levels were calculated using the ΔΔCt method, normalizing transcript levels to sigA signals. Data are shown as percentage relative to wild-type M. tuberculosis. (C) Immunoblots of cell lysates (CL) at 5 μg/well and culture filtrates (CF) at 10 μg/well of wild-type M. tuberculosis Erdman (WT) and espI ::Tn transformed with indicated plasmids grown in Sauton’s medium without Tween-80 for 5 days. Antibodies used are indicated. (D) Cytotoxicity assay in THP-1 cells infected with wild-type M. tuberculosis Erdman (Erd WT) (black bar) and espI ::Tn (grey bars) transformed with indicated plasmids at MOI of 5. Data represent the means and standard deviation of at least 4 independent experiments. The y-axis indicates cytotoxicity values relative to the uninfected control. Significance in difference compared to wild-type M. tuberculosis was calculated using Student’s t -test. *, p

    Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Transformation Assay, Cytotoxicity Assay, Infection, Standard Deviation

    19) Product Images from "Trappin-2/Elafin Modulate Innate Immune Responses of Human Endometrial Epithelial Cells to PolyI:C"

    Article Title: Trappin-2/Elafin Modulate Innate Immune Responses of Human Endometrial Epithelial Cells to PolyI:C

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035866

    Altered NF-κB phosphorylation and transcriptional activity and reduced RIG-I and MDA5 expression in polyI∶C-treated Ad/Tr-cells. Western blots ( A ) and their quantification ( B ) of phosphorylated p65 subunit of NF-κB (pNF-κB) as well as c-Jun (pc-Jun) and GAPDH proteins were performed from whole-cell extracts of Ad-cells that were either left untreated or treated with 25 µg/ml polyI∶C during indicated time points. Transcription activity ( C ) of NF-κB (NF-κB/Luc panel) and (AP-1/Luc panel) was assessed by luciferase reporter assay by transfecting HEC-1A cells with pgkβ-Gal and pNF-κB-Luc or pAP-1-Luc plasmids (total DNA 40 ng per well) (p) alone or together with 50 MOI of Ad/dl or Ad/Tr overnight, washing, allowing to rest for 4 h and stimulating with 25 µg/ml polyI∶C and 1 µg/ml LPS for 4 h. Then luciferase and β-galactosidase activities were determined in cell lysates and expressed as relative luciferase units using galactosidase plasmid as normalization control. Data are presented as mean ± SD and are representative of three experiments for NF-κB and two - for AP-1. ( D–F, left pane ls ) Total RNA was harvested and mRNA expression of RIG-I, MDA5, and TLR3 was determined by real-time quantitative RT-PCR at 6 h post treatment with polyI∶C 25 µg/ml. Values are normalized to a housekeeping gene 18S in the same sample and presented as fold induction over untreated cells. ( D–F, right panels ) Western blot analysis of RIG-I, MDA5, TLR3, and GAPDH protein was performed from whole-cell extracts of Ad-cells that were either treated or not with 25 µg/ml polyI∶C during indicated time points. The data are representative of at least two independent experiments performed in triplicate and are shown as the mean ± SD. Statistical analysis was performed using Student's t test with * representing significant difference between the groups, p
    Figure Legend Snippet: Altered NF-κB phosphorylation and transcriptional activity and reduced RIG-I and MDA5 expression in polyI∶C-treated Ad/Tr-cells. Western blots ( A ) and their quantification ( B ) of phosphorylated p65 subunit of NF-κB (pNF-κB) as well as c-Jun (pc-Jun) and GAPDH proteins were performed from whole-cell extracts of Ad-cells that were either left untreated or treated with 25 µg/ml polyI∶C during indicated time points. Transcription activity ( C ) of NF-κB (NF-κB/Luc panel) and (AP-1/Luc panel) was assessed by luciferase reporter assay by transfecting HEC-1A cells with pgkβ-Gal and pNF-κB-Luc or pAP-1-Luc plasmids (total DNA 40 ng per well) (p) alone or together with 50 MOI of Ad/dl or Ad/Tr overnight, washing, allowing to rest for 4 h and stimulating with 25 µg/ml polyI∶C and 1 µg/ml LPS for 4 h. Then luciferase and β-galactosidase activities were determined in cell lysates and expressed as relative luciferase units using galactosidase plasmid as normalization control. Data are presented as mean ± SD and are representative of three experiments for NF-κB and two - for AP-1. ( D–F, left pane ls ) Total RNA was harvested and mRNA expression of RIG-I, MDA5, and TLR3 was determined by real-time quantitative RT-PCR at 6 h post treatment with polyI∶C 25 µg/ml. Values are normalized to a housekeeping gene 18S in the same sample and presented as fold induction over untreated cells. ( D–F, right panels ) Western blot analysis of RIG-I, MDA5, TLR3, and GAPDH protein was performed from whole-cell extracts of Ad-cells that were either treated or not with 25 µg/ml polyI∶C during indicated time points. The data are representative of at least two independent experiments performed in triplicate and are shown as the mean ± SD. Statistical analysis was performed using Student's t test with * representing significant difference between the groups, p

    Techniques Used: Activity Assay, Expressing, Western Blot, Luciferase, Reporter Assay, Plasmid Preparation, Quantitative RT-PCR

    20) Product Images from "Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation"

    Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation

    Journal: Molecular and Cellular Neurosciences

    doi: 10.1016/j.mcn.2013.04.006

    Transfection with Wnt inhibitors does not prevent BDNF-induced c-Fos expression in cortical neurons. (A) GFP fluorescence, c-Fos immunoreactivity and DAPI fluorescence of representative cell bodies of cortical neurons expressing EV, Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ either alone, or in combination with BDNF. (B) Quantification of normalized nuclear c-Fos immunoreactivity per neuron for each treatment. *** p
    Figure Legend Snippet: Transfection with Wnt inhibitors does not prevent BDNF-induced c-Fos expression in cortical neurons. (A) GFP fluorescence, c-Fos immunoreactivity and DAPI fluorescence of representative cell bodies of cortical neurons expressing EV, Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ either alone, or in combination with BDNF. (B) Quantification of normalized nuclear c-Fos immunoreactivity per neuron for each treatment. *** p

    Techniques Used: Transfection, Expressing, Fluorescence

    21) Product Images from "An RS Motif within the Epstein-Barr Virus BLRF2 Tegument Protein Is Phosphorylated by SRPK2 and Is Important for Viral Replication"

    Article Title: An RS Motif within the Epstein-Barr Virus BLRF2 Tegument Protein Is Phosphorylated by SRPK2 and Is Important for Viral Replication

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053512

    BLRF2 rescues ORF52 null MHV68 replication, but the BLRF2-ARA mutant cannot. (A) Complementation assay measuring MHV68 virion release by quantitative PCR into supernatants of 293T cells co-transfected with replication defective MVH68 ORF52 null BAC and empty vector, wild-type FLAG-BLRF2, or FLAG-BLRF2-ARA mutant. Viral DNA was quantified four days post-transfection and the results shown are representative of two independent experiments performed in triplicate. Western blot with anti-flag antibody shows BLRF2-WT and –ARA expression levels.
    Figure Legend Snippet: BLRF2 rescues ORF52 null MHV68 replication, but the BLRF2-ARA mutant cannot. (A) Complementation assay measuring MHV68 virion release by quantitative PCR into supernatants of 293T cells co-transfected with replication defective MVH68 ORF52 null BAC and empty vector, wild-type FLAG-BLRF2, or FLAG-BLRF2-ARA mutant. Viral DNA was quantified four days post-transfection and the results shown are representative of two independent experiments performed in triplicate. Western blot with anti-flag antibody shows BLRF2-WT and –ARA expression levels.

    Techniques Used: Acetylene Reduction Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Transfection, BAC Assay, Plasmid Preparation, Western Blot, Expressing

    SRPK2 phosphorylates BLRF2. (A) Schematic of BLRF2 indicating the central dimerization domain (aa 66–122) and the C-terminal RS motif. ClustalW alignment of the C-termini of BLRF2 (aa 123 to 162) and its homologs from the rhesus and marmoset lymphocryptoviruses and murine gammaherpesvirus 68. Degree of conservation is shown at the bottom (* - identical, : - high, . - moderate). A conserved basic domain containing the putative RS motif is highlighted by the box. (B) GST pull-down of 293T cells transfected with GST-BLRF2 wild-type (WT) or GST-BLRF2 SRS-ARA mutant (ARA) in the presence or absence of GFP-SRPK2. Western blot analysis using anti-GFP (top panels) and anti-GST (lower panels) antibodies. Input lysates (1%) are shown in the left panels. (C) Western blots of transfected 293T whole cell lysates probed with anti-Phospho-SR antibody (left) and anti-GST antibody (right). (D) Western blots of GST pull-downs from 293T cells transfected with GST-BLRF2 wild-type or ARA mutant. Phosphorylated GST-BLRF2 is shown in the left panel with anti-Phospho-SR antibody and total GST-BLRF2 level is shown by anti-GST antibody (right panel). (E) Western blot of GST pull-downs of 293T cells transfected with GST-BLRF2 wild-type and ARA mutant along with increasing amounts of SRPK2-GFP. Phosphorylated protein is detected by anti-Phosho-SR antibody (top panels), anti-GFP antibody showed SRPK2-GFP (middle panels) and anti-GST antibody showed total BLRF2 (bottom panels). Input lysates (1%) are shown in the left panels.
    Figure Legend Snippet: SRPK2 phosphorylates BLRF2. (A) Schematic of BLRF2 indicating the central dimerization domain (aa 66–122) and the C-terminal RS motif. ClustalW alignment of the C-termini of BLRF2 (aa 123 to 162) and its homologs from the rhesus and marmoset lymphocryptoviruses and murine gammaherpesvirus 68. Degree of conservation is shown at the bottom (* - identical, : - high, . - moderate). A conserved basic domain containing the putative RS motif is highlighted by the box. (B) GST pull-down of 293T cells transfected with GST-BLRF2 wild-type (WT) or GST-BLRF2 SRS-ARA mutant (ARA) in the presence or absence of GFP-SRPK2. Western blot analysis using anti-GFP (top panels) and anti-GST (lower panels) antibodies. Input lysates (1%) are shown in the left panels. (C) Western blots of transfected 293T whole cell lysates probed with anti-Phospho-SR antibody (left) and anti-GST antibody (right). (D) Western blots of GST pull-downs from 293T cells transfected with GST-BLRF2 wild-type or ARA mutant. Phosphorylated GST-BLRF2 is shown in the left panel with anti-Phospho-SR antibody and total GST-BLRF2 level is shown by anti-GST antibody (right panel). (E) Western blot of GST pull-downs of 293T cells transfected with GST-BLRF2 wild-type and ARA mutant along with increasing amounts of SRPK2-GFP. Phosphorylated protein is detected by anti-Phosho-SR antibody (top panels), anti-GFP antibody showed SRPK2-GFP (middle panels) and anti-GST antibody showed total BLRF2 (bottom panels). Input lysates (1%) are shown in the left panels.

    Techniques Used: Transfection, Acetylene Reduction Assay, Mutagenesis, Western Blot

    The BLRF2 ARA mutant exhibits increased cytoplasmic localization compared to wild-type BLRF2. (A) Immunofluorescence microcopy showing BLRF2 localization in HeLa cells transfected with BLRF2 wild-type or ARA mutant. Cells were stained with anti-BLRF2 antibody (green) and Draq5 DNA staining (blue). Examples of predominantly nuclear (left panels), mixed nuclear and cytoplamsic (middle panels), and predominantly cytoplasmic staining (right panels) are shown. (B) Summary of immunofluorescence analysis described in (A). The percentage of cells observed with predominantly nuclear (N) (blue), cytoplasmic (C) (red) or mixed (N and C) (gray) BLRF2 staining are shown.
    Figure Legend Snippet: The BLRF2 ARA mutant exhibits increased cytoplasmic localization compared to wild-type BLRF2. (A) Immunofluorescence microcopy showing BLRF2 localization in HeLa cells transfected with BLRF2 wild-type or ARA mutant. Cells were stained with anti-BLRF2 antibody (green) and Draq5 DNA staining (blue). Examples of predominantly nuclear (left panels), mixed nuclear and cytoplamsic (middle panels), and predominantly cytoplasmic staining (right panels) are shown. (B) Summary of immunofluorescence analysis described in (A). The percentage of cells observed with predominantly nuclear (N) (blue), cytoplasmic (C) (red) or mixed (N and C) (gray) BLRF2 staining are shown.

    Techniques Used: Acetylene Reduction Assay, Mutagenesis, Immunofluorescence, Transfection, Staining

    22) Product Images from "Complement C5 Activation during Influenza A Infection in Mice Contributes to Neutrophil Recruitment and Lung Injury"

    Article Title: Complement C5 Activation during Influenza A Infection in Mice Contributes to Neutrophil Recruitment and Lung Injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064443

    Leukocyte recruitment and cfDNA levels in BALF after IAV infection upon OmCI and Zileuton treatment. C57BL/6J mice were infected intranasally with 10 4 PFU of Influenza A/WSN/33 H1N1, or received PBS intranasally (Mock group). The 5-LO inhibitor Zileuton (30 mg/kg) was given alone or in combination with OmCI. Mice received the treatment prior to the infection and daily until day 5 after infection, while vehicle group received PBS both, via i.p, Zileuton was given by oral gavage. At the sixth day after infection, mice were euthanized, BAL performed and lungs were harvested. A) Total number of leukocytes; B) number of neutrophils; C) cfDNA levels measured in BALF. Data are presented as Mean ± SEM. * and *** for p
    Figure Legend Snippet: Leukocyte recruitment and cfDNA levels in BALF after IAV infection upon OmCI and Zileuton treatment. C57BL/6J mice were infected intranasally with 10 4 PFU of Influenza A/WSN/33 H1N1, or received PBS intranasally (Mock group). The 5-LO inhibitor Zileuton (30 mg/kg) was given alone or in combination with OmCI. Mice received the treatment prior to the infection and daily until day 5 after infection, while vehicle group received PBS both, via i.p, Zileuton was given by oral gavage. At the sixth day after infection, mice were euthanized, BAL performed and lungs were harvested. A) Total number of leukocytes; B) number of neutrophils; C) cfDNA levels measured in BALF. Data are presented as Mean ± SEM. * and *** for p

    Techniques Used: Infection, Mouse Assay

    Effects of OmCI on cfDNA levels and number of dead cells in BALF after IAV infection. C57BL/6J mice were infected and assigned to treatment groups as in Figure 2 . At1, 3 and 6 days after infection, mice were euthanized and BAL was performed. A) Cell free DNA (cfDNA) levels were measured in BALF by the Quant-iT PicoGreen dsDNA quantification kit. At six days after infection, leukocytes recovered from airways of Mock, Vehicle and OmCI treated mice were analyzed for cellular death by Annexin V and PI incorporation. B) Total apoptotic leukocytes, Annexin V+ PI-, in BALF; C) Total necrotic or late apoptotic leukocytes, Annexin V+ PI+, in BALF. Data are presented as Mean ± SEM. ** and *** for p
    Figure Legend Snippet: Effects of OmCI on cfDNA levels and number of dead cells in BALF after IAV infection. C57BL/6J mice were infected and assigned to treatment groups as in Figure 2 . At1, 3 and 6 days after infection, mice were euthanized and BAL was performed. A) Cell free DNA (cfDNA) levels were measured in BALF by the Quant-iT PicoGreen dsDNA quantification kit. At six days after infection, leukocytes recovered from airways of Mock, Vehicle and OmCI treated mice were analyzed for cellular death by Annexin V and PI incorporation. B) Total apoptotic leukocytes, Annexin V+ PI-, in BALF; C) Total necrotic or late apoptotic leukocytes, Annexin V+ PI+, in BALF. Data are presented as Mean ± SEM. ** and *** for p

    Techniques Used: Infection, Mouse Assay

    23) Product Images from "A Novel Quantitative Fluorescent Reporter Assay for RAG Targets and RAG Activity"

    Article Title: A Novel Quantitative Fluorescent Reporter Assay for RAG Targets and RAG Activity

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2013.00110

    The GFPi reporter . (A) Linear representation of the GFPi reporter (left) and resulting structure following RAG activity (right); GFPi is constructed from a murine stem cell virus (MSCV) retroviral vector and the portion flanked by the long-terminal repeats (LTRs) is represented. From the 5′ to the 3′ LTR, the relevant segments are as follow: a 12-spacer RSS (light gray triangle), the GFP inverted complementary sequence (green box), a 23-spacer RSS (dark gray triangle), an internal ribosomal entry site (IRES), the RFP coding sequence (red box). Upon RAG-mediated GFP inversion formation of a coding joint (CJ) and signal joint (SJ) is indicated. The arrows represent the primers used to detect GFPi unrearranged (UNR) and rearranged (RR) CJ and SJ in (C) . (B) GFP/RFP flow cytometry contour plot analysis of a representative in vitro recombination assay. 293T cells were transfected with 10 μg DNA containing either only irrelevant DNA (no GFPi), 5 μg GFPi (GFPi) alone or together with 0.8 μg CMV-RAG1 and 0.7 μg CMV-RAG2 (GFPi + RAG). GFPi carried consensus 12 and 23-RSS. Upper plots are gated on live cells while lower plots are in addition gated on RFP + cells. Values represent the percentage of each cell population in the quadrant (upper plots) or in the GFP + gate (lower plots, n = 4). (C) Semi-quantitative PCR analysis of specific amplicons to detect the rearranged signal joint region of GFPi (GFPi-RR-SJ), or unrearranged GFPi (GFPi-UNR) performed with the indicated plasmid amounts isolated from sorted RFP + GFP + or RFP + GFP − 293T cells previously transfected with GFPi + RAG or the GFPi alone, respectively.
    Figure Legend Snippet: The GFPi reporter . (A) Linear representation of the GFPi reporter (left) and resulting structure following RAG activity (right); GFPi is constructed from a murine stem cell virus (MSCV) retroviral vector and the portion flanked by the long-terminal repeats (LTRs) is represented. From the 5′ to the 3′ LTR, the relevant segments are as follow: a 12-spacer RSS (light gray triangle), the GFP inverted complementary sequence (green box), a 23-spacer RSS (dark gray triangle), an internal ribosomal entry site (IRES), the RFP coding sequence (red box). Upon RAG-mediated GFP inversion formation of a coding joint (CJ) and signal joint (SJ) is indicated. The arrows represent the primers used to detect GFPi unrearranged (UNR) and rearranged (RR) CJ and SJ in (C) . (B) GFP/RFP flow cytometry contour plot analysis of a representative in vitro recombination assay. 293T cells were transfected with 10 μg DNA containing either only irrelevant DNA (no GFPi), 5 μg GFPi (GFPi) alone or together with 0.8 μg CMV-RAG1 and 0.7 μg CMV-RAG2 (GFPi + RAG). GFPi carried consensus 12 and 23-RSS. Upper plots are gated on live cells while lower plots are in addition gated on RFP + cells. Values represent the percentage of each cell population in the quadrant (upper plots) or in the GFP + gate (lower plots, n = 4). (C) Semi-quantitative PCR analysis of specific amplicons to detect the rearranged signal joint region of GFPi (GFPi-RR-SJ), or unrearranged GFPi (GFPi-UNR) performed with the indicated plasmid amounts isolated from sorted RFP + GFP + or RFP + GFP − 293T cells previously transfected with GFPi + RAG or the GFPi alone, respectively.

    Techniques Used: Activity Assay, Construct, Plasmid Preparation, Sequencing, Flow Cytometry, Cytometry, In Vitro, Recombination Assay, Transfection, Real-time Polymerase Chain Reaction, Isolation

    GFPi reports RAG transcription and nuclear translocation efficiencies . (A) Representative FACS analysis of 293T cells transfected with the GFPi reporter only (mock) or together with H2k-RAG1/2, H2k-RAG1 and H2k-RAG2ER TAM treated with vehicle alone or with 4-OHT, or CMV-RAG1/2. (B) Recombination efficiency determined as [(%GFP + in test) – (%GFP + in mock)]. Mean and standard deviation of at least six replicates and at least two independent experiments. (** – p
    Figure Legend Snippet: GFPi reports RAG transcription and nuclear translocation efficiencies . (A) Representative FACS analysis of 293T cells transfected with the GFPi reporter only (mock) or together with H2k-RAG1/2, H2k-RAG1 and H2k-RAG2ER TAM treated with vehicle alone or with 4-OHT, or CMV-RAG1/2. (B) Recombination efficiency determined as [(%GFP + in test) – (%GFP + in mock)]. Mean and standard deviation of at least six replicates and at least two independent experiments. (** – p

    Techniques Used: Translocation Assay, FACS, Transfection, Standard Deviation

    The GFPi assay efficiently ranks nucleotide sequences for their RSS functionality . GFPi variants carrying different 12 or 23-RSSs were tested in 293T cells transfected alone (mock) or co-transfected with either H2k-RAG1/2 (A) or CMV-RAG1/2 (B,D) . (A–D) GFPi 23-RSS variants Con23, ConPI, MI-conPI(4), MI-conPI(14,15), MI-conPI(19,20), MI, and MI-CAGnon were described previously (Cowell et al., 2004 ) and their nucleotide sequences are listed in Table 1 . (A,B) Representative FACS analysis gated on RFP + cells. Number are% of GFP + cells C ) Recombination efficiency defined as [(%GFP + in test) – (%GFP + in respective control)] (left and middle panel) At least six replicates from at least two independent experiments. Right panel shows the relative recombination coefficient ρ normalized to GFPi-Con. For MI-CAGnon, the less efficient RSS, ρ in the mock was
    Figure Legend Snippet: The GFPi assay efficiently ranks nucleotide sequences for their RSS functionality . GFPi variants carrying different 12 or 23-RSSs were tested in 293T cells transfected alone (mock) or co-transfected with either H2k-RAG1/2 (A) or CMV-RAG1/2 (B,D) . (A–D) GFPi 23-RSS variants Con23, ConPI, MI-conPI(4), MI-conPI(14,15), MI-conPI(19,20), MI, and MI-CAGnon were described previously (Cowell et al., 2004 ) and their nucleotide sequences are listed in Table 1 . (A,B) Representative FACS analysis gated on RFP + cells. Number are% of GFP + cells C ) Recombination efficiency defined as [(%GFP + in test) – (%GFP + in respective control)] (left and middle panel) At least six replicates from at least two independent experiments. Right panel shows the relative recombination coefficient ρ normalized to GFPi-Con. For MI-CAGnon, the less efficient RSS, ρ in the mock was

    Techniques Used: Transfection, FACS

    In vitro recombination assay using the GFPi-CFP variant . (A) Linear representation of the GFPi-CFP reporter. (B) The recombination of different RSSs pairs was determined upon 293T cells transfection with each GFPi-CFP variants together with the CMV-RAG1/2 expressing plasmids. The 23-RSS Con23 and MI-conPI(19,20), as well as the 12-RSS Con12 and LMO2 were as in Figure 5 . Wu-Con12 is a consensus 12-RSS defined by bio-informatics (Ramsden et al., 1994 ). The 3′Dδ3 is a physiological partner of LMO2 described in Dik et al. ( 2007 ). The specific pairs of RSS tested are indicated above the corresponding FACS plots and the sequences listed in Table 2 . The recombination efficiency defined as (% GFP + cells in test -% GFP + cells in control) is indicated in the upper part of each plot. The relative recombination coefficient ρ, when con12/con23 is 1, is indicated in the bottom part of each plot, in blue. four replicates of two independent experiments.
    Figure Legend Snippet: In vitro recombination assay using the GFPi-CFP variant . (A) Linear representation of the GFPi-CFP reporter. (B) The recombination of different RSSs pairs was determined upon 293T cells transfection with each GFPi-CFP variants together with the CMV-RAG1/2 expressing plasmids. The 23-RSS Con23 and MI-conPI(19,20), as well as the 12-RSS Con12 and LMO2 were as in Figure 5 . Wu-Con12 is a consensus 12-RSS defined by bio-informatics (Ramsden et al., 1994 ). The 3′Dδ3 is a physiological partner of LMO2 described in Dik et al. ( 2007 ). The specific pairs of RSS tested are indicated above the corresponding FACS plots and the sequences listed in Table 2 . The recombination efficiency defined as (% GFP + cells in test -% GFP + cells in control) is indicated in the upper part of each plot. The relative recombination coefficient ρ, when con12/con23 is 1, is indicated in the bottom part of each plot, in blue. four replicates of two independent experiments.

    Techniques Used: In Vitro, Recombination Assay, Variant Assay, Transfection, Expressing, FACS

    24) Product Images from "A novel EB-1/AIDA-1 isoform, AIDA-1c, interacts with the Cajal body protein coilin"

    Article Title: A novel EB-1/AIDA-1 isoform, AIDA-1c, interacts with the Cajal body protein coilin

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-6-23

    AIDA-1c expression is highest in neuronal tissue and is present in cancer lines . (A) A Multi-Tissue Northern blot (Clontech) was probed with an AIDA-1c specific probe. Re-probing of the same blot with a β-actin probe (bottom panel) is shown, verifying the presence of RNA in each lane. (B) (Upper panel) Nested PCR using AIDA-1c specific primers was conducted on cDNA obtained from a variety of cancer cell lines: SK-MEL-2 (melanoma), NIH:OVCAR-3 (ovarian), HCT-15 (colon), A-549 (non small cell lung carcinoma), MDA-MB-231 (breast), MCF-7 (breast). The positive control lane used AIDA-1c cDNA as the template. (Lower panel) HeLa expresses AIDA-1c. HeLa RNA was subjected to RT-PCR using an AIDA-1c specific primer. No product is observed in the reaction lacking reverse transcriptase (-RT).
    Figure Legend Snippet: AIDA-1c expression is highest in neuronal tissue and is present in cancer lines . (A) A Multi-Tissue Northern blot (Clontech) was probed with an AIDA-1c specific probe. Re-probing of the same blot with a β-actin probe (bottom panel) is shown, verifying the presence of RNA in each lane. (B) (Upper panel) Nested PCR using AIDA-1c specific primers was conducted on cDNA obtained from a variety of cancer cell lines: SK-MEL-2 (melanoma), NIH:OVCAR-3 (ovarian), HCT-15 (colon), A-549 (non small cell lung carcinoma), MDA-MB-231 (breast), MCF-7 (breast). The positive control lane used AIDA-1c cDNA as the template. (Lower panel) HeLa expresses AIDA-1c. HeLa RNA was subjected to RT-PCR using an AIDA-1c specific primer. No product is observed in the reaction lacking reverse transcriptase (-RT).

    Techniques Used: Expressing, Northern Blot, Nested PCR, Multiple Displacement Amplification, Positive Control, Reverse Transcription Polymerase Chain Reaction

    25) Product Images from "Pre-mRNA processing enhancer (PPE) elements from intronless genes play additional roles in mRNA biogenesis than do ones from intron-containing genes"

    Article Title: Pre-mRNA processing enhancer (PPE) elements from intronless genes play additional roles in mRNA biogenesis than do ones from intron-containing genes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki506

    The human c-Jun gene contains elements that enhance stabilization, 3′ end processing and cytoplasmic accumulation of intronless transcripts. ( A ) Autoradiogram of quantitative S1 nuclease mapping analysis of β-globin-like RNAs accumulated in the nucleus (N) and cytoplasm (C) 48 h after co-transfection of CV-1PD cells with the indicated plasmids and pRSV-Tori. The S1 nuclease mapping probes were the 5′ end-labeled ones shown in Figure 1B . The samples were analyzed as described in the legend to Figure 2A . The numbers below the lanes are mean ± SEM values of data obtained from three independent experiments similar to the one shown here. ( B ) Autoradiogram of quantitative S1 nuclease mapping analysis of the 3′ ends of the β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells. Portions of the RNA samples from the experiment shown in (A) were mapped with the 3′ end-labeled probe shown in Figure 1B . The numbers below the lanes are mean ± SEM values of data obtained from three independent experiments similar to the one shown here; they were calculated as described in the legend to Figure 2B . ( C ) The β-globin-like RNAs accumulated in the cytoplasm are unspliced. A portion of the RNA sample from the experiment shown in (A), lane 6, was reverse-transcribed and amplified by PCR as described in Figure 2C . Shown here is a photograph of an ethidium bromide-stained 1% agarose gel in which the PCR products were electrophoresed. The rightmost lane contains a 1 kilobase pair DNA ladder. ( D ) Autoradiogram of quantitative S1 nuclease mapping analysis showing that a 201 nt region of c-Jun is sufficient to enhance cytoplasmic accumulation of intronless β-globin-like transcripts. COS-M6 cells were transfected with the indicated plasmids and processed as described in (A).
    Figure Legend Snippet: The human c-Jun gene contains elements that enhance stabilization, 3′ end processing and cytoplasmic accumulation of intronless transcripts. ( A ) Autoradiogram of quantitative S1 nuclease mapping analysis of β-globin-like RNAs accumulated in the nucleus (N) and cytoplasm (C) 48 h after co-transfection of CV-1PD cells with the indicated plasmids and pRSV-Tori. The S1 nuclease mapping probes were the 5′ end-labeled ones shown in Figure 1B . The samples were analyzed as described in the legend to Figure 2A . The numbers below the lanes are mean ± SEM values of data obtained from three independent experiments similar to the one shown here. ( B ) Autoradiogram of quantitative S1 nuclease mapping analysis of the 3′ ends of the β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells. Portions of the RNA samples from the experiment shown in (A) were mapped with the 3′ end-labeled probe shown in Figure 1B . The numbers below the lanes are mean ± SEM values of data obtained from three independent experiments similar to the one shown here; they were calculated as described in the legend to Figure 2B . ( C ) The β-globin-like RNAs accumulated in the cytoplasm are unspliced. A portion of the RNA sample from the experiment shown in (A), lane 6, was reverse-transcribed and amplified by PCR as described in Figure 2C . Shown here is a photograph of an ethidium bromide-stained 1% agarose gel in which the PCR products were electrophoresed. The rightmost lane contains a 1 kilobase pair DNA ladder. ( D ) Autoradiogram of quantitative S1 nuclease mapping analysis showing that a 201 nt region of c-Jun is sufficient to enhance cytoplasmic accumulation of intronless β-globin-like transcripts. COS-M6 cells were transfected with the indicated plasmids and processed as described in (A).

    Techniques Used: Cotransfection, Labeling, Amplification, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Transfection

    Effects of the presence of a PRE, PPE, CTE or RRE in obviating the intron requirement for efficient 3′ end processing and cytoplasmic accumulation of human β-globin-like RNA. ( A ) Autoradiogram of quantitative S1 nuclease mapping analysis of the human β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells transfected with the plasmids shown in Figure 1A . CV-1PD cells were co-transfected with 2 μg of the indicated plasmid along with 1 μg of the SV40 T antigen-encoding plasmid pRSV-Tori. Nuclear (N) and cytoplasmic (C) RNAs were harvested 48 h later and analyzed by concurrent quantitative S1 nuclease mapping with the 5′ end-labeled β-globin and β-actin probes shown in Figure 1B . Transfection and RNA fractionation efficiencies were analyzed as described in Methods. The amount of β-globin-like RNA accumulated in the cytoplasm was internally normalized to the amount of cellular β-actin RNA present in the same sample. The numbers shown below the lanes are the percentages relative to the amount of β-globin-like RNA accumulated in the cytoplasm of cells transfected in parallel with β-β1(+)2(+) RNA, with normalization as well to the relative amounts of replicated β-globin plasmid DNA present in the nuclear fractions of those samples. These numbers are mean ± SEM values of data obtained from five independent experiments similar to the one shown here. ( B ) Autoradiogram of quantitative S1 nuclease mapping analysis of the 3′ ends of the β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells. The RNA samples from the experiment in panel A were analyzed with the 3′ end-labeled probe shown in Figure 1B . The numbers below the pairs of lanes are mean ± SEM values of the cleaved RNA (N + C) divided by the cleaved plus uncleaved RNA (N + C) times 100% of data obtained from three independent experiments similar to the one shown here. ( C ) The β-globin-like RNAs accumulated in the cytoplasm are unspliced. Portions of the cytoplasmic RNA samples from the experiment shown in (A) were reverse-transcribed and then amplified by PCR with the primers described in Methods (+RT, lanes 3, 6, 9, 12 and 15). As controls, PCR amplification reactions were performed on the RNA samples without prior reverse transcription (−RT, lanes 2, 5, 8, 11 and 14) and on the plasmid DNAs used in the transfections (DNA, lanes 4, 7, 10, 13 and 16). Shown here is a photograph of an ethidium bromide-stained 1% agarose gel in which the PCR products were electrophoresed. Lane 1 contained 1 kilobase pair ladder DNA as size markers.
    Figure Legend Snippet: Effects of the presence of a PRE, PPE, CTE or RRE in obviating the intron requirement for efficient 3′ end processing and cytoplasmic accumulation of human β-globin-like RNA. ( A ) Autoradiogram of quantitative S1 nuclease mapping analysis of the human β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells transfected with the plasmids shown in Figure 1A . CV-1PD cells were co-transfected with 2 μg of the indicated plasmid along with 1 μg of the SV40 T antigen-encoding plasmid pRSV-Tori. Nuclear (N) and cytoplasmic (C) RNAs were harvested 48 h later and analyzed by concurrent quantitative S1 nuclease mapping with the 5′ end-labeled β-globin and β-actin probes shown in Figure 1B . Transfection and RNA fractionation efficiencies were analyzed as described in Methods. The amount of β-globin-like RNA accumulated in the cytoplasm was internally normalized to the amount of cellular β-actin RNA present in the same sample. The numbers shown below the lanes are the percentages relative to the amount of β-globin-like RNA accumulated in the cytoplasm of cells transfected in parallel with β-β1(+)2(+) RNA, with normalization as well to the relative amounts of replicated β-globin plasmid DNA present in the nuclear fractions of those samples. These numbers are mean ± SEM values of data obtained from five independent experiments similar to the one shown here. ( B ) Autoradiogram of quantitative S1 nuclease mapping analysis of the 3′ ends of the β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells. The RNA samples from the experiment in panel A were analyzed with the 3′ end-labeled probe shown in Figure 1B . The numbers below the pairs of lanes are mean ± SEM values of the cleaved RNA (N + C) divided by the cleaved plus uncleaved RNA (N + C) times 100% of data obtained from three independent experiments similar to the one shown here. ( C ) The β-globin-like RNAs accumulated in the cytoplasm are unspliced. Portions of the cytoplasmic RNA samples from the experiment shown in (A) were reverse-transcribed and then amplified by PCR with the primers described in Methods (+RT, lanes 3, 6, 9, 12 and 15). As controls, PCR amplification reactions were performed on the RNA samples without prior reverse transcription (−RT, lanes 2, 5, 8, 11 and 14) and on the plasmid DNAs used in the transfections (DNA, lanes 4, 7, 10, 13 and 16). Shown here is a photograph of an ethidium bromide-stained 1% agarose gel in which the PCR products were electrophoresed. Lane 1 contained 1 kilobase pair ladder DNA as size markers.

    Techniques Used: Transfection, Plasmid Preparation, Labeling, Fractionation, Amplification, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis

    26) Product Images from "Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1"

    Article Title: Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074424

    Sp1 and Smad2 are specifically associated with the GDP-fucose transporter promoter. A. and B. ChIP analyses of the interactions of Sp1 and Smad2 with the GDP-Fuc transporter promoter upon TGF-β1 stimulation. HeLa cells were serum-starved and then incubated with human TGF-β1 for 8 h. Extracts for ChIP assays were prepared from the cells treated without (lane 2) or with (lanes 1 and 3) TGF-β1. ChIP was carried out with rabbit immunoglobulin (mock) (lane 1), anti-Sp1 (top), -Smad2 (middle) or -pSmad2 (bottom) antibodies. The resulting precipitates were amplified by PCR with the primers specific to the GDP-fucose transporter promoter region. The PCR products were analyzed on a 1% agarose gel (A). qPCR was performed with the precipitated DNA from the ChIP assay as above (B). Error bars represent standard deviation from three replicates. *, p
    Figure Legend Snippet: Sp1 and Smad2 are specifically associated with the GDP-fucose transporter promoter. A. and B. ChIP analyses of the interactions of Sp1 and Smad2 with the GDP-Fuc transporter promoter upon TGF-β1 stimulation. HeLa cells were serum-starved and then incubated with human TGF-β1 for 8 h. Extracts for ChIP assays were prepared from the cells treated without (lane 2) or with (lanes 1 and 3) TGF-β1. ChIP was carried out with rabbit immunoglobulin (mock) (lane 1), anti-Sp1 (top), -Smad2 (middle) or -pSmad2 (bottom) antibodies. The resulting precipitates were amplified by PCR with the primers specific to the GDP-fucose transporter promoter region. The PCR products were analyzed on a 1% agarose gel (A). qPCR was performed with the precipitated DNA from the ChIP assay as above (B). Error bars represent standard deviation from three replicates. *, p

    Techniques Used: Chromatin Immunoprecipitation, Incubation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation

    TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P
    Figure Legend Snippet: TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P

    Techniques Used: Expressing, Cell Culture, Recombinant, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR, Standard Deviation

    27) Product Images from "Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation"

    Article Title: Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075648

    Relative levels of the HCV 3’ X-associated proteins in Huh7.5, Clone B, and 293T cells. HCV 3’ X-associated proteins were fractionated on the 2D gel. The level of each protein (spot) was quantified using the PDQuest software, expressed in parts per million (ppm) and compared among Huh7.5, Clone B, and 293T cells (from left to right in each histogram). The identity of each protein revealed by MASS was given on the top of the histogram along with the SwissProt accession number in parenthesis and the standard spot (SSP) number at the bottom of the histogram.
    Figure Legend Snippet: Relative levels of the HCV 3’ X-associated proteins in Huh7.5, Clone B, and 293T cells. HCV 3’ X-associated proteins were fractionated on the 2D gel. The level of each protein (spot) was quantified using the PDQuest software, expressed in parts per million (ppm) and compared among Huh7.5, Clone B, and 293T cells (from left to right in each histogram). The identity of each protein revealed by MASS was given on the top of the histogram along with the SwissProt accession number in parenthesis and the standard spot (SSP) number at the bottom of the histogram.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Software

    Scheme of the RNA affinity chromatography-2D/MASS strategy. Cell extracts were prepared from 293T, Huh7.5, and Clone B cells and were added to streptavidin agarose beads that were pre-conjugated with biotinylated HCV 3’X RNA. The beads were extensively washed to remove unbound proteins, and the proteins that either directly or indirectly bound to RNA were then eluted and analyzed by 2D/MASS.
    Figure Legend Snippet: Scheme of the RNA affinity chromatography-2D/MASS strategy. Cell extracts were prepared from 293T, Huh7.5, and Clone B cells and were added to streptavidin agarose beads that were pre-conjugated with biotinylated HCV 3’X RNA. The beads were extensively washed to remove unbound proteins, and the proteins that either directly or indirectly bound to RNA were then eluted and analyzed by 2D/MASS.

    Techniques Used: Affinity Chromatography

    28) Product Images from "Mitochondrial DAMPs Increase Endothelial Permeability through Neutrophil Dependent and Independent Pathways"

    Article Title: Mitochondrial DAMPs Increase Endothelial Permeability through Neutrophil Dependent and Independent Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059989

    mtDNA and MTD induce permeability in EA calls. (A) mtDNA-induced endothelial permeability changes were evaluated by ECIS as described in Figure 1 . EA cells were seeded onto ECIS cultureware and 25 or 50 µg/mL mtDNA was applied with or without 2–3×10 5 human PMN. (B) In addition to the conditions in 2A, 5 µM ODN TTAGGG (a specific inhibitor of TLR9) was applied to chambers where indicated. Two or three wells were used per reaction, mean and SD values are shown. (C) EA cells were pre-treated with 10 or 50 µM Chloroquine then MTD (×1/25) were applied. Two or three wells are used per reaction, mean and SD values are shown. For 2A, B and C the conditions were the same as described in Figure 1 . All experiments were repeated at least three times. (D) Unlike EA or HPAEC, HMVEC have very slow and limited responses to stimulation.
    Figure Legend Snippet: mtDNA and MTD induce permeability in EA calls. (A) mtDNA-induced endothelial permeability changes were evaluated by ECIS as described in Figure 1 . EA cells were seeded onto ECIS cultureware and 25 or 50 µg/mL mtDNA was applied with or without 2–3×10 5 human PMN. (B) In addition to the conditions in 2A, 5 µM ODN TTAGGG (a specific inhibitor of TLR9) was applied to chambers where indicated. Two or three wells were used per reaction, mean and SD values are shown. (C) EA cells were pre-treated with 10 or 50 µM Chloroquine then MTD (×1/25) were applied. Two or three wells are used per reaction, mean and SD values are shown. For 2A, B and C the conditions were the same as described in Figure 1 . All experiments were repeated at least three times. (D) Unlike EA or HPAEC, HMVEC have very slow and limited responses to stimulation.

    Techniques Used: Permeability, Electric Cell-substrate Impedance Sensing

    Externally applied mtDNA localizes to endosomes. HPAEC were reacted with mtDNA labeled with Alexa-488, FM4-64 (a marker for endosomes) and NucBlue (a marker for nuclei). After 4 hr of incubation, cells were examined for dye localization by confocal microscopy. A. nuclei, B. endosomes, and C. mtDNA were separately detected. Co-localization of mtDNA (Alexa 488) and the endosomal marker (FM4-64) is shown in D. Experiments were repeated at least three times.
    Figure Legend Snippet: Externally applied mtDNA localizes to endosomes. HPAEC were reacted with mtDNA labeled with Alexa-488, FM4-64 (a marker for endosomes) and NucBlue (a marker for nuclei). After 4 hr of incubation, cells were examined for dye localization by confocal microscopy. A. nuclei, B. endosomes, and C. mtDNA were separately detected. Co-localization of mtDNA (Alexa 488) and the endosomal marker (FM4-64) is shown in D. Experiments were repeated at least three times.

    Techniques Used: Labeling, Marker, Incubation, Confocal Microscopy

    29) Product Images from "Convergent transcription induces transcriptional gene silencing in fission yeast and mammalian cells"

    Article Title: Convergent transcription induces transcriptional gene silencing in fission yeast and mammalian cells

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2392

    in vitro and in vivo analysis of nuclear dicer activity (a) Western blot analysis of dicer and Hsp-70 from whole cell extract (WE) versus nuclear extract (NE) with or without dicer immunodepletion (NE-dicer). Hsp70 was only detectable in WE confirming purity of NE. (b) In vitro transcription. NE dependent in vitro transcription of CTγACT1 Ex4 (390 nt RNA) and vector alone plus control run off template (yielding 363 nt RNA). Following the transcription reaction, RNA was isolated and fractionated. The long RNA fraction was treated with S1 (single strand specific), V1 (double strand specific) nucleases. Lower panel shows that dicer depleted NE still yields CT derived dsRNA. (c) In vitro transcription. Fractionation of small RNAs isolated from templates as indicated ( Supplementary Fig. 4 ). S denotes single promoter construct making just a sense transcript. Only CT and CTT yield detectible siRNAs (denoted by arrow) but not in dicer depleted NE. (d) qRT-PCR. Immunoselection of dsRNA from CTγACT1 or V transfected Hela cells using J2 antibody. Sense and antisense transcripts from CTγACT1 or endogenous GAPDH were monitored by qRT/PCR using strand specific RT primers. RT-PCR values in are based on average values ± SD from at least three independent biological experiments. * indicates statistical significance (p
    Figure Legend Snippet: in vitro and in vivo analysis of nuclear dicer activity (a) Western blot analysis of dicer and Hsp-70 from whole cell extract (WE) versus nuclear extract (NE) with or without dicer immunodepletion (NE-dicer). Hsp70 was only detectable in WE confirming purity of NE. (b) In vitro transcription. NE dependent in vitro transcription of CTγACT1 Ex4 (390 nt RNA) and vector alone plus control run off template (yielding 363 nt RNA). Following the transcription reaction, RNA was isolated and fractionated. The long RNA fraction was treated with S1 (single strand specific), V1 (double strand specific) nucleases. Lower panel shows that dicer depleted NE still yields CT derived dsRNA. (c) In vitro transcription. Fractionation of small RNAs isolated from templates as indicated ( Supplementary Fig. 4 ). S denotes single promoter construct making just a sense transcript. Only CT and CTT yield detectible siRNAs (denoted by arrow) but not in dicer depleted NE. (d) qRT-PCR. Immunoselection of dsRNA from CTγACT1 or V transfected Hela cells using J2 antibody. Sense and antisense transcripts from CTγACT1 or endogenous GAPDH were monitored by qRT/PCR using strand specific RT primers. RT-PCR values in are based on average values ± SD from at least three independent biological experiments. * indicates statistical significance (p

    Techniques Used: In Vitro, In Vivo, Activity Assay, Western Blot, Plasmid Preparation, Isolation, Derivative Assay, Fractionation, Construct, Quantitative RT-PCR, Transfection, Reverse Transcription Polymerase Chain Reaction

    CT ura4 plasmids induce TGS of endogenous ura4 in S. pombe (a) CT ura4 plasmid diagram showing endogenous ura4 with indicated positions of ChIP probes on endogenous ura4 as indicated. (b) qRT-PCR. CT ura4 plasmids containing either ura4 ORF or promoter sequences induce selective reduction of endogenous ura4 but not act1 mRNA levels as measured by qRT-PCR using oligodT primer for cDNA synthesis. Empty CT vector (V) was used as normalizing control. Induction period of CT nmt1 promoters by growth in EMM was overnight (as for all experiments except for (c,d)). (c,d) qRT-PCR of nascent ura4 RNA (using RT primer across PAS) or ura4 mRNA (using oligodT RT primer) following induction of CT for three days. (e,f) ChIP analysis using Pol II specific antibody on chromatin isolated for CTura4prom or CTura4ORF transformed S. pombe with PCR primer pairs as indicated in (a). (g,h) ChIP analysis using histone H3K9me3 specific antibody as in (e,f) All RT-PCR and ChIP values are based on average values ± SD from at least three independent biological experiments. * indicates statistical significance (p
    Figure Legend Snippet: CT ura4 plasmids induce TGS of endogenous ura4 in S. pombe (a) CT ura4 plasmid diagram showing endogenous ura4 with indicated positions of ChIP probes on endogenous ura4 as indicated. (b) qRT-PCR. CT ura4 plasmids containing either ura4 ORF or promoter sequences induce selective reduction of endogenous ura4 but not act1 mRNA levels as measured by qRT-PCR using oligodT primer for cDNA synthesis. Empty CT vector (V) was used as normalizing control. Induction period of CT nmt1 promoters by growth in EMM was overnight (as for all experiments except for (c,d)). (c,d) qRT-PCR of nascent ura4 RNA (using RT primer across PAS) or ura4 mRNA (using oligodT RT primer) following induction of CT for three days. (e,f) ChIP analysis using Pol II specific antibody on chromatin isolated for CTura4prom or CTura4ORF transformed S. pombe with PCR primer pairs as indicated in (a). (g,h) ChIP analysis using histone H3K9me3 specific antibody as in (e,f) All RT-PCR and ChIP values are based on average values ± SD from at least three independent biological experiments. * indicates statistical significance (p

    Techniques Used: Plasmid Preparation, Chromatin Immunoprecipitation, Quantitative RT-PCR, Isolation, Transformation Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Integrated CT constructs promote endogenous ura4 TGS in trans (a,b) qRT-PCR. iCTura4ORF transformed S. pombe shows reduced levels of endogenous ura4 nascent RNA(a) and mRNA(b) based on qRT-PCR analysis. Diagram shows integrated CT chromosome with position of RT primer used to detect nascent RNA (not cleaved at PAS). Also shown, nmt1 promoters (red arrows) and endogenous ura4 . (c,d) ChIP analysis. Endogenous ura4 is subjected to trans TGS from iCTura4ORF as judged by Pol II and H3K9me3 ChIP analysis. (e,f) qRT-PCR. Induction of iCTcdc10 and iCTrad21 integrated into S. pombe causes a selective reduction in endogenous cdc10 and rad21 mRNA levels based on qRT-PCR analysis. All RT-PCR and ChIP values in a-f) are based on average values ± SD from at least three independent biological experiments. * indicates statistical significance (p
    Figure Legend Snippet: Integrated CT constructs promote endogenous ura4 TGS in trans (a,b) qRT-PCR. iCTura4ORF transformed S. pombe shows reduced levels of endogenous ura4 nascent RNA(a) and mRNA(b) based on qRT-PCR analysis. Diagram shows integrated CT chromosome with position of RT primer used to detect nascent RNA (not cleaved at PAS). Also shown, nmt1 promoters (red arrows) and endogenous ura4 . (c,d) ChIP analysis. Endogenous ura4 is subjected to trans TGS from iCTura4ORF as judged by Pol II and H3K9me3 ChIP analysis. (e,f) qRT-PCR. Induction of iCTcdc10 and iCTrad21 integrated into S. pombe causes a selective reduction in endogenous cdc10 and rad21 mRNA levels based on qRT-PCR analysis. All RT-PCR and ChIP values in a-f) are based on average values ± SD from at least three independent biological experiments. * indicates statistical significance (p

    Techniques Used: Construct, Quantitative RT-PCR, Transformation Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Transfection of mammalian CTura4 plasmid induces γ-ACT1 TGS (a) Diagram of CT γACT1 containing γACT1 cDNA (exons 2-6). (b) Pol II ChIP analysis of γ- ACT1 or GAPDH control using amplicons specific to endogenous gene as shown on gene diagram below. Chromatin was isolated from HeLa cells transiently transfected with CT vector alone (V), CTγACT1 or untransfected (UN). (c) qRT-PCR. Measurement of mRNA levels using oligodT primed qRT-PCR on RNA from HeLa cells transfected as in (b). (d) H3K9me3 antibody ChIP as in (b). (e) Western blot analysis and quantitation of γ-actin protein levels compared to tubulin and OAS1 on total protein isolated from HeLa cells transfected as in (b) for 1-3 days. (f) qRT-PCR. Effect of CTγACT1 transfection of ES cells, wt or ΔDCR1 on nascent transcript levels from γ- ACT1 versus GAPDH . (g) Ago2 ChIP on chromatin from V or CTγACT1 transfected HeLa cells using specific amplicons for endogenous γ- ACT1 or GAPDH . All RT-PCR and ChIP values in b, c, d, f and g are based on average values ± SD from at least three independent biological experiments. * indicates statistical significance (p
    Figure Legend Snippet: Transfection of mammalian CTura4 plasmid induces γ-ACT1 TGS (a) Diagram of CT γACT1 containing γACT1 cDNA (exons 2-6). (b) Pol II ChIP analysis of γ- ACT1 or GAPDH control using amplicons specific to endogenous gene as shown on gene diagram below. Chromatin was isolated from HeLa cells transiently transfected with CT vector alone (V), CTγACT1 or untransfected (UN). (c) qRT-PCR. Measurement of mRNA levels using oligodT primed qRT-PCR on RNA from HeLa cells transfected as in (b). (d) H3K9me3 antibody ChIP as in (b). (e) Western blot analysis and quantitation of γ-actin protein levels compared to tubulin and OAS1 on total protein isolated from HeLa cells transfected as in (b) for 1-3 days. (f) qRT-PCR. Effect of CTγACT1 transfection of ES cells, wt or ΔDCR1 on nascent transcript levels from γ- ACT1 versus GAPDH . (g) Ago2 ChIP on chromatin from V or CTγACT1 transfected HeLa cells using specific amplicons for endogenous γ- ACT1 or GAPDH . All RT-PCR and ChIP values in b, c, d, f and g are based on average values ± SD from at least three independent biological experiments. * indicates statistical significance (p

    Techniques Used: Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Isolation, Quantitative RT-PCR, Western Blot, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction

    30) Product Images from "Identification of a Tissue-Selective Heat Shock Response Regulatory Network"

    Article Title: Identification of a Tissue-Selective Heat Shock Response Regulatory Network

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003466

    Validation of tissue-selective effects using small molecules and mutants. A) Incubation with 100 µM MG132, a small molecule inhibitor of the proteasome, but not DMSO alone, causes tissue-selective induction of the phsp70::gfp reporter in the intestine and spermatheca (arrows), similar to RNAi knockdown of proteasomal subunits. The scale bar corresponds to 200 µm. Asterisks denote only autofluorescence. B) Mutations in T24H7.2, sgt-1, cyn-11, and unc-45 cause induction of the HSR in whole worms measured using qRT-PCR. C) Mutation of T24H7.2 , but not unc-45 , causes induction in the intestine relative to N2 control animals, measured by qRT-PCR analysis of hsp70 in dissected intestinal tissue. Averages shown are from at least two biological replicates.
    Figure Legend Snippet: Validation of tissue-selective effects using small molecules and mutants. A) Incubation with 100 µM MG132, a small molecule inhibitor of the proteasome, but not DMSO alone, causes tissue-selective induction of the phsp70::gfp reporter in the intestine and spermatheca (arrows), similar to RNAi knockdown of proteasomal subunits. The scale bar corresponds to 200 µm. Asterisks denote only autofluorescence. B) Mutations in T24H7.2, sgt-1, cyn-11, and unc-45 cause induction of the HSR in whole worms measured using qRT-PCR. C) Mutation of T24H7.2 , but not unc-45 , causes induction in the intestine relative to N2 control animals, measured by qRT-PCR analysis of hsp70 in dissected intestinal tissue. Averages shown are from at least two biological replicates.

    Techniques Used: Incubation, Quantitative RT-PCR, Mutagenesis

    Genome-wide RNAi screen for HSR regulators. (A–D) Nomarski and fluorescent images corresponding to the phsp70::gfp reporter strain. (A) Control animals show little reporter expression and only faint autofluorescence of the intestine. (B) Reporter induction in animals exposed to heat shock at 33°C for 1 hour. (C) RNAi knockdown of hsf-1 decreases induction of the reporter by heat shock. (D) RNAi knockdown of hsp-1 causes constitutive induction of the reporter in the absence of heat shock. The scale bar corresponds to 100 µm. (E–F) Quantitation of the effects on endogenous hsp70 and hsp-16.2 genes using qRT-PCR. (E) HSR positive regulators normalized to the heat shocked empty vector control. (F) HSR negative regulators normalized to empty vector control. Averages are from at least three biological replicates and error bars represent SEM.
    Figure Legend Snippet: Genome-wide RNAi screen for HSR regulators. (A–D) Nomarski and fluorescent images corresponding to the phsp70::gfp reporter strain. (A) Control animals show little reporter expression and only faint autofluorescence of the intestine. (B) Reporter induction in animals exposed to heat shock at 33°C for 1 hour. (C) RNAi knockdown of hsf-1 decreases induction of the reporter by heat shock. (D) RNAi knockdown of hsp-1 causes constitutive induction of the reporter in the absence of heat shock. The scale bar corresponds to 100 µm. (E–F) Quantitation of the effects on endogenous hsp70 and hsp-16.2 genes using qRT-PCR. (E) HSR positive regulators normalized to the heat shocked empty vector control. (F) HSR negative regulators normalized to empty vector control. Averages are from at least three biological replicates and error bars represent SEM.

    Techniques Used: Genome Wide, Expressing, Quantitation Assay, Quantitative RT-PCR, Plasmid Preparation

    31) Product Images from "Rai14 (Retinoic Acid Induced Protein 14) Is Involved in Regulating F-Actin Dynamics at the Ectoplasmic Specialization in the Rat Testis*"

    Article Title: Rai14 (Retinoic Acid Induced Protein 14) Is Involved in Regulating F-Actin Dynamics at the Ectoplasmic Specialization in the Rat Testis*

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060656

    Rai14 is an actin-binding protein in the rat testis. ( A ) A study by RT-PCR to confirm the expression of Rai14 in adult rat testis, Sertoli cells (SC, isolated from 20-day-old rat testes and cultured for 4-day), and germ cells (GC, isolated from adult rat testes and cultured for 16 hr). ( B ) Immunoblotting also confirmed the expression of Rai14 in the rat testis, Sertoli and germ cells, and the relative expression of Rai14 in SC vs. GC was shown in the histogram with n = 3 experiments in which the relative expression level of Rai14 in the testis was arbitrarily set at 1 so that the relative expression level between these samples can be compared. ( C ) The specificity of the anti-Rai14 antibody ( Table 1 ) was assessed by immunoblotting using lysates of GC (20 µg protein). ( D ) Using the specific anti-Rai14 antibody, Rai14 was shown to be an actin-binding protein by co-immunoprecipitation (Co-IP); however, Rai14 did not structurally interact with any of the BTB-associated proteins including several actin-binding and regulatory proteins ( e.g ., Arp3, drebrin E, Eps8) and vimentin (an intermediate filament-based constituent protein). However, Rai14 was found to structurally interact with an actin cross-linking protein palladin which is known to be involved in conferring actin filament bundles in other mammalian cells [48] . ( E ) Rai14 (red) was also shown to be an actin-binding protein by dual-labeled immunofluorescence analysis in which it co-localized with F-actin (green) in Sertoli cells. Cell nuclei (blue) were visualized by DAPI. Scale ba = 20 µm, which applies to all other micrographs.
    Figure Legend Snippet: Rai14 is an actin-binding protein in the rat testis. ( A ) A study by RT-PCR to confirm the expression of Rai14 in adult rat testis, Sertoli cells (SC, isolated from 20-day-old rat testes and cultured for 4-day), and germ cells (GC, isolated from adult rat testes and cultured for 16 hr). ( B ) Immunoblotting also confirmed the expression of Rai14 in the rat testis, Sertoli and germ cells, and the relative expression of Rai14 in SC vs. GC was shown in the histogram with n = 3 experiments in which the relative expression level of Rai14 in the testis was arbitrarily set at 1 so that the relative expression level between these samples can be compared. ( C ) The specificity of the anti-Rai14 antibody ( Table 1 ) was assessed by immunoblotting using lysates of GC (20 µg protein). ( D ) Using the specific anti-Rai14 antibody, Rai14 was shown to be an actin-binding protein by co-immunoprecipitation (Co-IP); however, Rai14 did not structurally interact with any of the BTB-associated proteins including several actin-binding and regulatory proteins ( e.g ., Arp3, drebrin E, Eps8) and vimentin (an intermediate filament-based constituent protein). However, Rai14 was found to structurally interact with an actin cross-linking protein palladin which is known to be involved in conferring actin filament bundles in other mammalian cells [48] . ( E ) Rai14 (red) was also shown to be an actin-binding protein by dual-labeled immunofluorescence analysis in which it co-localized with F-actin (green) in Sertoli cells. Cell nuclei (blue) were visualized by DAPI. Scale ba = 20 µm, which applies to all other micrographs.

    Techniques Used: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Cell Culture, Immunoprecipitation, Co-Immunoprecipitation Assay, Labeling, Immunofluorescence

    Knockdown of Rai14 in the Sertoli cell epithelium with an established TJ-permeability barrier in vitro by RNAi disrupts actin filament organization and the TJ barrier. (A) Sertoli cells cultured alone on Matrigel-coated 12-well dishes for 2-day with an established TJ-permeability barrier were transfected with Rai14 siRNA duplexes (Rai14 RNAi) versus non-targeting control duplexes (Ctrl RNAi) at 100 nM using Ribojuice transfection medium for 24 hr, thereafter, cells were washed twice and cultured in F12/DMEM for 12 hr to allow recovery. Thereafter, cells were transfected again under the same conditions for another 24 hr. Thereafter, cells were rinsed with fresh F12/DMEM and cultured for an additional 12 hr before termination, and used to prepare lysates for immunoblotting using antibodies against several BTB-associated constituent or regulatory proteins. A knockdown of Rai14 by ∼50% was noted in which the control was arbitrarily set at 1 against which statistical comparison was performed (B) without any apparent off-target effects (A). The findings shown herein are the results of 3 independent experiments excluding pilot experiments which were used to establish optimal experimental conditions, such as different concentrations of siRNA duplexes and Ribojuice. It was noted that we achieved only ∼50–60% knockdown of Rai14 in several pilot experiments, unlike other target genes [ e.g ., Scribble, β1-integrin, and P-glycoprotein] wherein we could silence the target gene expression by as much as ∼70–90%. **, P
    Figure Legend Snippet: Knockdown of Rai14 in the Sertoli cell epithelium with an established TJ-permeability barrier in vitro by RNAi disrupts actin filament organization and the TJ barrier. (A) Sertoli cells cultured alone on Matrigel-coated 12-well dishes for 2-day with an established TJ-permeability barrier were transfected with Rai14 siRNA duplexes (Rai14 RNAi) versus non-targeting control duplexes (Ctrl RNAi) at 100 nM using Ribojuice transfection medium for 24 hr, thereafter, cells were washed twice and cultured in F12/DMEM for 12 hr to allow recovery. Thereafter, cells were transfected again under the same conditions for another 24 hr. Thereafter, cells were rinsed with fresh F12/DMEM and cultured for an additional 12 hr before termination, and used to prepare lysates for immunoblotting using antibodies against several BTB-associated constituent or regulatory proteins. A knockdown of Rai14 by ∼50% was noted in which the control was arbitrarily set at 1 against which statistical comparison was performed (B) without any apparent off-target effects (A). The findings shown herein are the results of 3 independent experiments excluding pilot experiments which were used to establish optimal experimental conditions, such as different concentrations of siRNA duplexes and Ribojuice. It was noted that we achieved only ∼50–60% knockdown of Rai14 in several pilot experiments, unlike other target genes [ e.g ., Scribble, β1-integrin, and P-glycoprotein] wherein we could silence the target gene expression by as much as ∼70–90%. **, P

    Techniques Used: Permeability, In Vitro, Cell Culture, Transfection, Expressing

    Stage-specific expression of Rai14 and its association with the F-actin-rich ectoplasmic specialization (ES) in the seminiferous epithelium of adult rat testes. Dual-labeled immunofluorescence analysis was performed using frozen cross-sections of testes from adult rat testes to examine co-localization of Rai14 (red) and F-actin (green) in the seminiferous epithelium. At stage VI, Rai14 was weakly detected at the apical ES at the Sertoli-spermatid interface, and its localization to the BTB was also weakly detectable. However, at stage VII-early stage VIII, the expression of Rai14 was the strongest. Rai14 was intensely localized to the apical ES, most abundantly at the front-end of the spermatid head, and co-localized with F-actin at the site (“yellow” in merged image of the apical ES), and its localization at the BTB remained not clearly visible. At stage VIII, Rai14 expression at the apical ES was still considerably strong, but it no longer restricted to the front-end of the spermatid head, instead, Rai14 was scattered around the spermatid head at the apical ES, but not tightly co-localized with the F-actin when compared to late stage VII tubule. In late stage VIII tubules, Rai14 also considerably expressed near the basement membrane (annotated by “white” broken line), consistent with its localization at the BTB (see “white” arrowheads), but not tightly co-localized with F-actin. At stage XI-XII, Rai14 remained localized to the apical ES, but it also shifted to the front-end of the spermatid head, partially co-localized with F-actin. “Yellow” and “green” boxed areas were magnified and shown in corresponding micrographs to better illustrate the localization and/or co-localization of Rai14 and/or Rai14/F-actin at the apical ES. Scale bar = 50 µm or 10 µm in the micrograph or inset, respectively, which apply to all other micrographs and insets.
    Figure Legend Snippet: Stage-specific expression of Rai14 and its association with the F-actin-rich ectoplasmic specialization (ES) in the seminiferous epithelium of adult rat testes. Dual-labeled immunofluorescence analysis was performed using frozen cross-sections of testes from adult rat testes to examine co-localization of Rai14 (red) and F-actin (green) in the seminiferous epithelium. At stage VI, Rai14 was weakly detected at the apical ES at the Sertoli-spermatid interface, and its localization to the BTB was also weakly detectable. However, at stage VII-early stage VIII, the expression of Rai14 was the strongest. Rai14 was intensely localized to the apical ES, most abundantly at the front-end of the spermatid head, and co-localized with F-actin at the site (“yellow” in merged image of the apical ES), and its localization at the BTB remained not clearly visible. At stage VIII, Rai14 expression at the apical ES was still considerably strong, but it no longer restricted to the front-end of the spermatid head, instead, Rai14 was scattered around the spermatid head at the apical ES, but not tightly co-localized with the F-actin when compared to late stage VII tubule. In late stage VIII tubules, Rai14 also considerably expressed near the basement membrane (annotated by “white” broken line), consistent with its localization at the BTB (see “white” arrowheads), but not tightly co-localized with F-actin. At stage XI-XII, Rai14 remained localized to the apical ES, but it also shifted to the front-end of the spermatid head, partially co-localized with F-actin. “Yellow” and “green” boxed areas were magnified and shown in corresponding micrographs to better illustrate the localization and/or co-localization of Rai14 and/or Rai14/F-actin at the apical ES. Scale bar = 50 µm or 10 µm in the micrograph or inset, respectively, which apply to all other micrographs and insets.

    Techniques Used: Expressing, Labeling, Immunofluorescence

    32) Product Images from "KSHV PAN RNA Associates with Demethylases UTX and JMJD3 to Activate Lytic Replication through a Physical Interaction with the Virus Genome"

    Article Title: KSHV PAN RNA Associates with Demethylases UTX and JMJD3 to Activate Lytic Replication through a Physical Interaction with the Virus Genome

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002680

    Overexpression of K-Rta cannot complement BAC36CRΔPAN. (A) BACmid containing cell lines were transfected with a K-Rta expression plasmid and supernatant virus DNA was measured 4 days post transfection. The experiment was repeated 3 times. Error bars are the standard deviation from the mean. (B) Trans expression of K-Rta activates viral promoters in BAC36CRΔPAN containing cells and expression is enhanced in the presence of PAN RNA. BAC36CRΔPAN containing cells were transfected with K-Rta with or without the cotransfection of the PAN RNA expression plasmid and qPCR analysis was performed to measure mRNA accumulation for several viral encoded genes.
    Figure Legend Snippet: Overexpression of K-Rta cannot complement BAC36CRΔPAN. (A) BACmid containing cell lines were transfected with a K-Rta expression plasmid and supernatant virus DNA was measured 4 days post transfection. The experiment was repeated 3 times. Error bars are the standard deviation from the mean. (B) Trans expression of K-Rta activates viral promoters in BAC36CRΔPAN containing cells and expression is enhanced in the presence of PAN RNA. BAC36CRΔPAN containing cells were transfected with K-Rta with or without the cotransfection of the PAN RNA expression plasmid and qPCR analysis was performed to measure mRNA accumulation for several viral encoded genes.

    Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation, Standard Deviation, Cotransfection, RNA Expression, Real-time Polymerase Chain Reaction

    JMJD3 and UTX demethylases interact with KSHV DNA in the presence of PAN RNA expression. Cell lines containing BAC36CR or BAC36CRΔPAN were transfected with a K-Rta expression plasmid and ChIP assays were performed 3 days post transfection. Immunoprecipitations were performed using antibodies specific for JMJD3, UTX, K-Rta or an isotype specific antibody control. PCR primers specific for the ORF50 promoter or ORF45 were used to amplify immunoprecipitated DNA. Panel BAC36CRΔPAN+PAN: cells were transfected with both a K-Rta and PAN RNA expression plasmid.
    Figure Legend Snippet: JMJD3 and UTX demethylases interact with KSHV DNA in the presence of PAN RNA expression. Cell lines containing BAC36CR or BAC36CRΔPAN were transfected with a K-Rta expression plasmid and ChIP assays were performed 3 days post transfection. Immunoprecipitations were performed using antibodies specific for JMJD3, UTX, K-Rta or an isotype specific antibody control. PCR primers specific for the ORF50 promoter or ORF45 were used to amplify immunoprecipitated DNA. Panel BAC36CRΔPAN+PAN: cells were transfected with both a K-Rta and PAN RNA expression plasmid.

    Techniques Used: RNA Expression, Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation

    PAN RNA interacts with demethylases and the histone methyltransferase MLL2. (A) TREx/BCBL-1 Rta cells were treated with DOX and RNA CLIP assays were performed 3 days post treatment. PAN RNA-protein complexes were immunoprecipitated using anti-JMJD3, anti-UTX, anti-MML2 or isotype control antibodies. PCR primers were used to amplify (after RT) PAN RNA, ORF45 RNA or U1 RNA. Also shown is PCR amplification without a reverse transcriptase reaction (PAN no RT). (B) PAN RNA expression leads to a relative decrease in the H3K27me3 mark on the ORF50 promoter. BAC36CR or BAC36CRΔPAN containing cells were transfected with either a K-Rta expression plasmid and/or a plasmid expressing PAN RNA. ChIP assays were performed using anti-H3K27me3 specific antibody. Immunoprecipitated DNA was analyzed by qPCR normalized to input DNA. Data is reported as fold decrease compared to BAC36CR untreated samples. Error bars are the standard deviation of the mean from three separate experiments.
    Figure Legend Snippet: PAN RNA interacts with demethylases and the histone methyltransferase MLL2. (A) TREx/BCBL-1 Rta cells were treated with DOX and RNA CLIP assays were performed 3 days post treatment. PAN RNA-protein complexes were immunoprecipitated using anti-JMJD3, anti-UTX, anti-MML2 or isotype control antibodies. PCR primers were used to amplify (after RT) PAN RNA, ORF45 RNA or U1 RNA. Also shown is PCR amplification without a reverse transcriptase reaction (PAN no RT). (B) PAN RNA expression leads to a relative decrease in the H3K27me3 mark on the ORF50 promoter. BAC36CR or BAC36CRΔPAN containing cells were transfected with either a K-Rta expression plasmid and/or a plasmid expressing PAN RNA. ChIP assays were performed using anti-H3K27me3 specific antibody. Immunoprecipitated DNA was analyzed by qPCR normalized to input DNA. Data is reported as fold decrease compared to BAC36CR untreated samples. Error bars are the standard deviation of the mean from three separate experiments.

    Techniques Used: Cross-linking Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Amplification, RNA Expression, Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

    PAN RNA physically interacts with the ORF50 promoter. (A) PAN RNA is enriched 30-fold by ChIRP assay. PAN RNA or LacZ specific biotinylated oligonucleotides were used to enrich PAN RNA. Recovered RNA or RNA from the depleted lysate (post ChIRP) was measured by qPCR. (B) TREx/BCBL-1 Rta cells were treated with DOX and 3 days post treatment ChIRP assays were performed. Tiling biotinylated oligonucleotides were used that hybridized to either PAN RNA (20 oligonucleotides) or control LacZ RNA (20 oligonucleotides). Pulled down DNA that was occupied by RNA was amplified using primers specific for the ORF50 promoter region or K6 ORF coding sequence.
    Figure Legend Snippet: PAN RNA physically interacts with the ORF50 promoter. (A) PAN RNA is enriched 30-fold by ChIRP assay. PAN RNA or LacZ specific biotinylated oligonucleotides were used to enrich PAN RNA. Recovered RNA or RNA from the depleted lysate (post ChIRP) was measured by qPCR. (B) TREx/BCBL-1 Rta cells were treated with DOX and 3 days post treatment ChIRP assays were performed. Tiling biotinylated oligonucleotides were used that hybridized to either PAN RNA (20 oligonucleotides) or control LacZ RNA (20 oligonucleotides). Pulled down DNA that was occupied by RNA was amplified using primers specific for the ORF50 promoter region or K6 ORF coding sequence.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Sequencing

    Generation of a recombinant BACmid with the PAN RNA locus deleted, BAC36CRΔPAN. (A) The BAC36CR template was used to insert the GalK-KanR cassette such that 634 nts of the PAN RNA gene was removed from the genome. (B) The GalK-KanR cassette was removed by homologous recombination and reverse selection. (C) BAC36CRΔPAN was generated by removal of the GalK-KanR cassette and the putative polyadenlyation signal downstream of the original PAN RNA and K7 genes was preserved. (D) Ethidium bromide stained agarose gel and Southern blot of BAC36, BAC36CR and BAC36CRΔPAN DNA cleaved with BamHI showing the removal of part of the PAN RNA locus.
    Figure Legend Snippet: Generation of a recombinant BACmid with the PAN RNA locus deleted, BAC36CRΔPAN. (A) The BAC36CR template was used to insert the GalK-KanR cassette such that 634 nts of the PAN RNA gene was removed from the genome. (B) The GalK-KanR cassette was removed by homologous recombination and reverse selection. (C) BAC36CRΔPAN was generated by removal of the GalK-KanR cassette and the putative polyadenlyation signal downstream of the original PAN RNA and K7 genes was preserved. (D) Ethidium bromide stained agarose gel and Southern blot of BAC36, BAC36CR and BAC36CRΔPAN DNA cleaved with BamHI showing the removal of part of the PAN RNA locus.

    Techniques Used: Recombinant, Homologous Recombination, Selection, Generated, Staining, Agarose Gel Electrophoresis, Southern Blot

    Deletion of the duplicated region within BAC36. (A) Duplicated genomic region is located between two terminal repeat sequences of the BAC36 genome (B) The duplicated ORFs 18-K5 were removed by insertion of a GalK-KanR cassette using oligonucleotides homologous to regions outside of the duplicated region. (C) Replacement of the GalK-KanR cassette in the BAC36 genome to yield the recombinant BACmid BAC36CR where the entire duplicated regions was removed. Shown is the DNA sequence after removal of the cassette (D) Ethidium bromide stained gel and Southern blot of BAC36 and BAC36CR DNA cleaved with BamHI and hybridized with either a probe specific for the GalK-KanR cassette (GalK probe) or the PAN RNA locus (PAN probe). Lanes: 1, MW marker; 2, BAC36, 3, BAC36+GalK-KanR cassette; 4, BAC36CR. Arrows indicated the PAN RNA locus in the unique long region of the genome and the duplicated PAN RNA locus located between the terminal repeats.
    Figure Legend Snippet: Deletion of the duplicated region within BAC36. (A) Duplicated genomic region is located between two terminal repeat sequences of the BAC36 genome (B) The duplicated ORFs 18-K5 were removed by insertion of a GalK-KanR cassette using oligonucleotides homologous to regions outside of the duplicated region. (C) Replacement of the GalK-KanR cassette in the BAC36 genome to yield the recombinant BACmid BAC36CR where the entire duplicated regions was removed. Shown is the DNA sequence after removal of the cassette (D) Ethidium bromide stained gel and Southern blot of BAC36 and BAC36CR DNA cleaved with BamHI and hybridized with either a probe specific for the GalK-KanR cassette (GalK probe) or the PAN RNA locus (PAN probe). Lanes: 1, MW marker; 2, BAC36, 3, BAC36+GalK-KanR cassette; 4, BAC36CR. Arrows indicated the PAN RNA locus in the unique long region of the genome and the duplicated PAN RNA locus located between the terminal repeats.

    Techniques Used: Recombinant, Sequencing, Staining, Southern Blot, Marker

    33) Product Images from "KSHV PAN RNA Associates with Demethylases UTX and JMJD3 to Activate Lytic Replication through a Physical Interaction with the Virus Genome"

    Article Title: KSHV PAN RNA Associates with Demethylases UTX and JMJD3 to Activate Lytic Replication through a Physical Interaction with the Virus Genome

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002680

    Overexpression of K-Rta cannot complement BAC36CRΔPAN. (A) BACmid containing cell lines were transfected with a K-Rta expression plasmid and supernatant virus DNA was measured 4 days post transfection. The experiment was repeated 3 times. Error bars are the standard deviation from the mean. (B) Trans expression of K-Rta activates viral promoters in BAC36CRΔPAN containing cells and expression is enhanced in the presence of PAN RNA. BAC36CRΔPAN containing cells were transfected with K-Rta with or without the cotransfection of the PAN RNA expression plasmid and qPCR analysis was performed to measure mRNA accumulation for several viral encoded genes.
    Figure Legend Snippet: Overexpression of K-Rta cannot complement BAC36CRΔPAN. (A) BACmid containing cell lines were transfected with a K-Rta expression plasmid and supernatant virus DNA was measured 4 days post transfection. The experiment was repeated 3 times. Error bars are the standard deviation from the mean. (B) Trans expression of K-Rta activates viral promoters in BAC36CRΔPAN containing cells and expression is enhanced in the presence of PAN RNA. BAC36CRΔPAN containing cells were transfected with K-Rta with or without the cotransfection of the PAN RNA expression plasmid and qPCR analysis was performed to measure mRNA accumulation for several viral encoded genes.

    Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation, Standard Deviation, Cotransfection, RNA Expression, Real-time Polymerase Chain Reaction

    JMJD3 and UTX demethylases interact with KSHV DNA in the presence of PAN RNA expression. Cell lines containing BAC36CR or BAC36CRΔPAN were transfected with a K-Rta expression plasmid and ChIP assays were performed 3 days post transfection. Immunoprecipitations were performed using antibodies specific for JMJD3, UTX, K-Rta or an isotype specific antibody control. PCR primers specific for the ORF50 promoter or ORF45 were used to amplify immunoprecipitated DNA. Panel BAC36CRΔPAN+PAN: cells were transfected with both a K-Rta and PAN RNA expression plasmid.
    Figure Legend Snippet: JMJD3 and UTX demethylases interact with KSHV DNA in the presence of PAN RNA expression. Cell lines containing BAC36CR or BAC36CRΔPAN were transfected with a K-Rta expression plasmid and ChIP assays were performed 3 days post transfection. Immunoprecipitations were performed using antibodies specific for JMJD3, UTX, K-Rta or an isotype specific antibody control. PCR primers specific for the ORF50 promoter or ORF45 were used to amplify immunoprecipitated DNA. Panel BAC36CRΔPAN+PAN: cells were transfected with both a K-Rta and PAN RNA expression plasmid.

    Techniques Used: RNA Expression, Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation

    PAN RNA interacts with demethylases and the histone methyltransferase MLL2. (A) TREx/BCBL-1 Rta cells were treated with DOX and RNA CLIP assays were performed 3 days post treatment. PAN RNA-protein complexes were immunoprecipitated using anti-JMJD3, anti-UTX, anti-MML2 or isotype control antibodies. PCR primers were used to amplify (after RT) PAN RNA, ORF45 RNA or U1 RNA. Also shown is PCR amplification without a reverse transcriptase reaction (PAN no RT). (B) PAN RNA expression leads to a relative decrease in the H3K27me3 mark on the ORF50 promoter. BAC36CR or BAC36CRΔPAN containing cells were transfected with either a K-Rta expression plasmid and/or a plasmid expressing PAN RNA. ChIP assays were performed using anti-H3K27me3 specific antibody. Immunoprecipitated DNA was analyzed by qPCR normalized to input DNA. Data is reported as fold decrease compared to BAC36CR untreated samples. Error bars are the standard deviation of the mean from three separate experiments.
    Figure Legend Snippet: PAN RNA interacts with demethylases and the histone methyltransferase MLL2. (A) TREx/BCBL-1 Rta cells were treated with DOX and RNA CLIP assays were performed 3 days post treatment. PAN RNA-protein complexes were immunoprecipitated using anti-JMJD3, anti-UTX, anti-MML2 or isotype control antibodies. PCR primers were used to amplify (after RT) PAN RNA, ORF45 RNA or U1 RNA. Also shown is PCR amplification without a reverse transcriptase reaction (PAN no RT). (B) PAN RNA expression leads to a relative decrease in the H3K27me3 mark on the ORF50 promoter. BAC36CR or BAC36CRΔPAN containing cells were transfected with either a K-Rta expression plasmid and/or a plasmid expressing PAN RNA. ChIP assays were performed using anti-H3K27me3 specific antibody. Immunoprecipitated DNA was analyzed by qPCR normalized to input DNA. Data is reported as fold decrease compared to BAC36CR untreated samples. Error bars are the standard deviation of the mean from three separate experiments.

    Techniques Used: Cross-linking Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Amplification, RNA Expression, Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

    PAN RNA physically interacts with the ORF50 promoter. (A) PAN RNA is enriched 30-fold by ChIRP assay. PAN RNA or LacZ specific biotinylated oligonucleotides were used to enrich PAN RNA. Recovered RNA or RNA from the depleted lysate (post ChIRP) was measured by qPCR. (B) TREx/BCBL-1 Rta cells were treated with DOX and 3 days post treatment ChIRP assays were performed. Tiling biotinylated oligonucleotides were used that hybridized to either PAN RNA (20 oligonucleotides) or control LacZ RNA (20 oligonucleotides). Pulled down DNA that was occupied by RNA was amplified using primers specific for the ORF50 promoter region or K6 ORF coding sequence.
    Figure Legend Snippet: PAN RNA physically interacts with the ORF50 promoter. (A) PAN RNA is enriched 30-fold by ChIRP assay. PAN RNA or LacZ specific biotinylated oligonucleotides were used to enrich PAN RNA. Recovered RNA or RNA from the depleted lysate (post ChIRP) was measured by qPCR. (B) TREx/BCBL-1 Rta cells were treated with DOX and 3 days post treatment ChIRP assays were performed. Tiling biotinylated oligonucleotides were used that hybridized to either PAN RNA (20 oligonucleotides) or control LacZ RNA (20 oligonucleotides). Pulled down DNA that was occupied by RNA was amplified using primers specific for the ORF50 promoter region or K6 ORF coding sequence.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Sequencing

    Generation of a recombinant BACmid with the PAN RNA locus deleted, BAC36CRΔPAN. (A) The BAC36CR template was used to insert the GalK-KanR cassette such that 634 nts of the PAN RNA gene was removed from the genome. (B) The GalK-KanR cassette was removed by homologous recombination and reverse selection. (C) BAC36CRΔPAN was generated by removal of the GalK-KanR cassette and the putative polyadenlyation signal downstream of the original PAN RNA and K7 genes was preserved. (D) Ethidium bromide stained agarose gel and Southern blot of BAC36, BAC36CR and BAC36CRΔPAN DNA cleaved with BamHI showing the removal of part of the PAN RNA locus.
    Figure Legend Snippet: Generation of a recombinant BACmid with the PAN RNA locus deleted, BAC36CRΔPAN. (A) The BAC36CR template was used to insert the GalK-KanR cassette such that 634 nts of the PAN RNA gene was removed from the genome. (B) The GalK-KanR cassette was removed by homologous recombination and reverse selection. (C) BAC36CRΔPAN was generated by removal of the GalK-KanR cassette and the putative polyadenlyation signal downstream of the original PAN RNA and K7 genes was preserved. (D) Ethidium bromide stained agarose gel and Southern blot of BAC36, BAC36CR and BAC36CRΔPAN DNA cleaved with BamHI showing the removal of part of the PAN RNA locus.

    Techniques Used: Recombinant, Homologous Recombination, Selection, Generated, Staining, Agarose Gel Electrophoresis, Southern Blot

    Deletion of the duplicated region within BAC36. (A) Duplicated genomic region is located between two terminal repeat sequences of the BAC36 genome (B) The duplicated ORFs 18-K5 were removed by insertion of a GalK-KanR cassette using oligonucleotides homologous to regions outside of the duplicated region. (C) Replacement of the GalK-KanR cassette in the BAC36 genome to yield the recombinant BACmid BAC36CR where the entire duplicated regions was removed. Shown is the DNA sequence after removal of the cassette (D) Ethidium bromide stained gel and Southern blot of BAC36 and BAC36CR DNA cleaved with BamHI and hybridized with either a probe specific for the GalK-KanR cassette (GalK probe) or the PAN RNA locus (PAN probe). Lanes: 1, MW marker; 2, BAC36, 3, BAC36+GalK-KanR cassette; 4, BAC36CR. Arrows indicated the PAN RNA locus in the unique long region of the genome and the duplicated PAN RNA locus located between the terminal repeats.
    Figure Legend Snippet: Deletion of the duplicated region within BAC36. (A) Duplicated genomic region is located between two terminal repeat sequences of the BAC36 genome (B) The duplicated ORFs 18-K5 were removed by insertion of a GalK-KanR cassette using oligonucleotides homologous to regions outside of the duplicated region. (C) Replacement of the GalK-KanR cassette in the BAC36 genome to yield the recombinant BACmid BAC36CR where the entire duplicated regions was removed. Shown is the DNA sequence after removal of the cassette (D) Ethidium bromide stained gel and Southern blot of BAC36 and BAC36CR DNA cleaved with BamHI and hybridized with either a probe specific for the GalK-KanR cassette (GalK probe) or the PAN RNA locus (PAN probe). Lanes: 1, MW marker; 2, BAC36, 3, BAC36+GalK-KanR cassette; 4, BAC36CR. Arrows indicated the PAN RNA locus in the unique long region of the genome and the duplicated PAN RNA locus located between the terminal repeats.

    Techniques Used: Recombinant, Sequencing, Staining, Southern Blot, Marker

    34) Product Images from "Modulation of Human Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus US Proteins"

    Article Title: Modulation of Human Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus US Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036163

    Generation of US-transduced MSC. (A) HCMV US2, US3, US6 and US11 cDNA sequences were cloned into the pMSCVneo plasmid between EcoRI and XhoI or Sal-I. US genes were driven by the MSCV LTR promoter while the Neomycin resistance marker gene (NeoR) was under the control of an internal PGK promoter. (B) Total RNA was extracted from each of the transduced and untransduced MSC populations and, after reverse transcription, cDNAs were obtained and amplified using specific primers for each of the US genes, NeoR and B-Actin. (C) Light microscope image at 10× original magnification of the different MSC populations showing similar morphology during cell culture. Images were captured with an Olympus IX-71 microscope.
    Figure Legend Snippet: Generation of US-transduced MSC. (A) HCMV US2, US3, US6 and US11 cDNA sequences were cloned into the pMSCVneo plasmid between EcoRI and XhoI or Sal-I. US genes were driven by the MSCV LTR promoter while the Neomycin resistance marker gene (NeoR) was under the control of an internal PGK promoter. (B) Total RNA was extracted from each of the transduced and untransduced MSC populations and, after reverse transcription, cDNAs were obtained and amplified using specific primers for each of the US genes, NeoR and B-Actin. (C) Light microscope image at 10× original magnification of the different MSC populations showing similar morphology during cell culture. Images were captured with an Olympus IX-71 microscope.

    Techniques Used: Clone Assay, Plasmid Preparation, Marker, Amplification, Light Microscopy, Cell Culture, Microscopy

    35) Product Images from "Detection of low-level promoter activity within open reading frame sequences of Escherichia coli"

    Article Title: Detection of low-level promoter activity within open reading frame sequences of Escherichia coli

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki928

    Detection of antisense transcripts by RT–PCR and northern analysis. ( A ) RT–PCR was performed with primer sets listed in Supplementary Table S1 and with (plus) and without (minus) RT. The products were analyzed by electrophoresis on 2% agarose gels. Lanes 1 and 2, ygeH antisense transcript (expected size, 124 bp); lanes 3 and 4, rhsE antisense transcript (expected size, 95 bp); lanes 5 and 6, yfjN antisense transcript (expected size, 92 bp); lanes 7 and 8, yiaO antisense transcript (expected size, 87 bp); lanes 9 and 10, yjcE antisense transcript (expected size, 103 bp); lanes 11 and 12, ydiM antisense transcript (expected size, 114 bp); lanes 13 and 14, ecpD antisense transcript (expected size, 119 bp); lanes 15 and 16, topA antisense transcript (expected size, 101 bp); lanes 17 and 18, yehI antisense transcript (expected size, 108 bp); lane M for 50 bp ladder size marker and lanes 19 and 20, RyjC RNA (expected size, 77 bp). The lack of correlation between the RT–PCR signal and β-galactosidase activity may be due to differences in the hybridization efficiencies for the primer pairs used. ( B ) Total RNA separated on 1% agarose gels and transferred to nylon membranes was probed with primers to the antisense strands of rhsE , yfjN , yiaO and yjcE as well as to the 77 nt antisense RNA RyjC. RNA molecular weight markers were run with each set of samples for direct estimation of RNA transcript length, but the RNA marker lane for only one of the panels is shown.
    Figure Legend Snippet: Detection of antisense transcripts by RT–PCR and northern analysis. ( A ) RT–PCR was performed with primer sets listed in Supplementary Table S1 and with (plus) and without (minus) RT. The products were analyzed by electrophoresis on 2% agarose gels. Lanes 1 and 2, ygeH antisense transcript (expected size, 124 bp); lanes 3 and 4, rhsE antisense transcript (expected size, 95 bp); lanes 5 and 6, yfjN antisense transcript (expected size, 92 bp); lanes 7 and 8, yiaO antisense transcript (expected size, 87 bp); lanes 9 and 10, yjcE antisense transcript (expected size, 103 bp); lanes 11 and 12, ydiM antisense transcript (expected size, 114 bp); lanes 13 and 14, ecpD antisense transcript (expected size, 119 bp); lanes 15 and 16, topA antisense transcript (expected size, 101 bp); lanes 17 and 18, yehI antisense transcript (expected size, 108 bp); lane M for 50 bp ladder size marker and lanes 19 and 20, RyjC RNA (expected size, 77 bp). The lack of correlation between the RT–PCR signal and β-galactosidase activity may be due to differences in the hybridization efficiencies for the primer pairs used. ( B ) Total RNA separated on 1% agarose gels and transferred to nylon membranes was probed with primers to the antisense strands of rhsE , yfjN , yiaO and yjcE as well as to the 77 nt antisense RNA RyjC. RNA molecular weight markers were run with each set of samples for direct estimation of RNA transcript length, but the RNA marker lane for only one of the panels is shown.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot, Electrophoresis, Marker, Activity Assay, Hybridization, Molecular Weight

    36) Product Images from "Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen"

    Article Title: Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002751

    The course of infection with L. major GFP + in BALB/c mice vaccinated with different modalities. BALB/c mice were immunized in the right footpad with L. tarentolae CPA/CPB/EGFP (G1, rLive/rLive); primed with DNA PpSP15 and boosted with L. tarentolae CPA/CPB/EGFP and DNA PpSP15 (G2, DNA/rLive+DNA); primed and boosted with both L. tarentolae CPA/CPB/EGFP and DNA PpSP15 (G3, DNA+rLive/DNA+rLive); injected with PBS as control (G4), vaccination and boosting with VR1020-SP15 (G5, DNA/DNA); G6: vaccination and boosting with L. tarentolae EGFP+ (G6, control Live/Live). All animals were challenged with stationary phase L. major (2×10 6 /mice) plus SGH (0.5 pair) in the left footpad except for G1 and G6, which received only L. major . A) The footpad swelling represents the Mean±SD of 12 mice per group; Asterisks indicate statistical significance (Mann Whitney U test, * p
    Figure Legend Snippet: The course of infection with L. major GFP + in BALB/c mice vaccinated with different modalities. BALB/c mice were immunized in the right footpad with L. tarentolae CPA/CPB/EGFP (G1, rLive/rLive); primed with DNA PpSP15 and boosted with L. tarentolae CPA/CPB/EGFP and DNA PpSP15 (G2, DNA/rLive+DNA); primed and boosted with both L. tarentolae CPA/CPB/EGFP and DNA PpSP15 (G3, DNA+rLive/DNA+rLive); injected with PBS as control (G4), vaccination and boosting with VR1020-SP15 (G5, DNA/DNA); G6: vaccination and boosting with L. tarentolae EGFP+ (G6, control Live/Live). All animals were challenged with stationary phase L. major (2×10 6 /mice) plus SGH (0.5 pair) in the left footpad except for G1 and G6, which received only L. major . A) The footpad swelling represents the Mean±SD of 12 mice per group; Asterisks indicate statistical significance (Mann Whitney U test, * p

    Techniques Used: Infection, Mouse Assay, Injection, MANN-WHITNEY

    The course of infection with L. major GFP+ in C57BL/6 mice vaccinated with different modalities. C57BL/6 mice were immunized in the right footpad with L. tarentolae CPA/CPB/EGFP (G1; rLive/rLive); primed with DNA PpSP15 and boosted with L. tarentolae CPA/CPB/EGFP and DNA PpSP15 (G2; DNA/rLive+DNA); primed and boosted with L. tarentolae CPA/CPB/EGFP and DNA PpSP15 (G3; DNA+rLive/DNA+rLive); injected with PBS as control (G4), vaccination and boosting with VR1020-SP15 (G5, DNA/DNA); G6: vaccination and boosting with L. tarentolae EGFP+ (G6, control Live/Live). All animals were challenged with stationary phase L. major (2×10 6 /mice) plus SGH (0.5 pair) in the left footpad except for G1 and G6 which only received L. major . A) The footpad swelling represents the Mean±SD of 12 mice per group. Asterisks indicate statistical significance (Mann Whitney U test, p
    Figure Legend Snippet: The course of infection with L. major GFP+ in C57BL/6 mice vaccinated with different modalities. C57BL/6 mice were immunized in the right footpad with L. tarentolae CPA/CPB/EGFP (G1; rLive/rLive); primed with DNA PpSP15 and boosted with L. tarentolae CPA/CPB/EGFP and DNA PpSP15 (G2; DNA/rLive+DNA); primed and boosted with L. tarentolae CPA/CPB/EGFP and DNA PpSP15 (G3; DNA+rLive/DNA+rLive); injected with PBS as control (G4), vaccination and boosting with VR1020-SP15 (G5, DNA/DNA); G6: vaccination and boosting with L. tarentolae EGFP+ (G6, control Live/Live). All animals were challenged with stationary phase L. major (2×10 6 /mice) plus SGH (0.5 pair) in the left footpad except for G1 and G6 which only received L. major . A) The footpad swelling represents the Mean±SD of 12 mice per group. Asterisks indicate statistical significance (Mann Whitney U test, p

    Techniques Used: Infection, Mouse Assay, Injection, MANN-WHITNEY

    37) Product Images from "Target labelling for the detection and profiling of microRNAs expressed in CNS tissue using microarrays"

    Article Title: Target labelling for the detection and profiling of microRNAs expressed in CNS tissue using microarrays

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-6-47

    Heat map of signal intensities from miRNAs that were similarly identified by both types of labelling techniques .(†) Represents miRNAs that are no longer listed in the miRNA registry however present on the custom microarray. (‡) Represents miRNA-like elements from references 41 and 47. We scored TaqMan detected miRNA probes as present using the following criteria. Ath-miR-159a, cel-lin-4, and cel-miR-2 were used as the negative controls (Ct ~30–35). The Ct values used to determine +, ++, +++ expression patterns are as follows: +++ – 15–18; ++ – 18 – 21; + – 21–27;
    Figure Legend Snippet: Heat map of signal intensities from miRNAs that were similarly identified by both types of labelling techniques .(†) Represents miRNAs that are no longer listed in the miRNA registry however present on the custom microarray. (‡) Represents miRNA-like elements from references 41 and 47. We scored TaqMan detected miRNA probes as present using the following criteria. Ath-miR-159a, cel-lin-4, and cel-miR-2 were used as the negative controls (Ct ~30–35). The Ct values used to determine +, ++, +++ expression patterns are as follows: +++ – 15–18; ++ – 18 – 21; + – 21–27;

    Techniques Used: Microarray, Expressing

    Detection range of miRNAs by the linear-amplification strategy . A synthetic miRNA (miR-124a) was titrated, linear amplified, and hybridized to the arrays. Signal to background ratio and specificity of binding of the miRNA is shown. The amount of miRNA that can be reliably amplified and detected by the microarray is denoted with an asterisk.
    Figure Legend Snippet: Detection range of miRNAs by the linear-amplification strategy . A synthetic miRNA (miR-124a) was titrated, linear amplified, and hybridized to the arrays. Signal to background ratio and specificity of binding of the miRNA is shown. The amount of miRNA that can be reliably amplified and detected by the microarray is denoted with an asterisk.

    Techniques Used: Amplification, Binding Assay, Microarray

    Venn diagram to illustrate miRNAs detected by TaqMan ® MiRNA Assay quantification and miRNA microarray analysis.
    Figure Legend Snippet: Venn diagram to illustrate miRNAs detected by TaqMan ® MiRNA Assay quantification and miRNA microarray analysis.

    Techniques Used: TaqMan microRNA Assay, Microarray

    Specificity of the complementary miRNA microarray probes . Synthetic miRNA targets were hybridized to arrays containing probes that had increasing number of nucleotide mismatches at either the 3' or 5' ends.
    Figure Legend Snippet: Specificity of the complementary miRNA microarray probes . Synthetic miRNA targets were hybridized to arrays containing probes that had increasing number of nucleotide mismatches at either the 3' or 5' ends.

    Techniques Used: Microarray

    38) Product Images from "Nuclear Translocation and Regulation of Intranuclear Distribution of Cytoplasmic Poly(A)-Binding Protein Are Distinct Processes Mediated by Two Epstein Barr Virus Proteins"

    Article Title: Nuclear Translocation and Regulation of Intranuclear Distribution of Cytoplasmic Poly(A)-Binding Protein Are Distinct Processes Mediated by Two Epstein Barr Virus Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092593

    ZEBRA and BGLF5 decrease levels of GFP mRNA and protein; one point mutant of ZEBRA does not inhibit GFP expression. (A) 293 cells were transfected with pHD1013, or vectors expressing GFP, ZEBRA, or FLAG-BGLF5. RNA extracts were prepared 45 h after transfection. Real-time RT-PCR analysis was performed using primers specific for GFP and 18S rRNA. Real time RT-PCR values for GFP were normalized to 18S rRNA values. Error bars were derived from variation in values obtained from technical replicates performed in triplicate. (B) 293 cells were co-transfected with GFP and vector, ZEBRA, Z(S186A), Z(S186E), or Z(N182K). Cell extracts were prepared 45 h after transfection and analyzed by SDS-page. Immunoblots were probed with antibody specific for GFP, ZEBRA, and β-actin. The levels of GFP were quantified by densitometry and normalized to levels of β-actin.
    Figure Legend Snippet: ZEBRA and BGLF5 decrease levels of GFP mRNA and protein; one point mutant of ZEBRA does not inhibit GFP expression. (A) 293 cells were transfected with pHD1013, or vectors expressing GFP, ZEBRA, or FLAG-BGLF5. RNA extracts were prepared 45 h after transfection. Real-time RT-PCR analysis was performed using primers specific for GFP and 18S rRNA. Real time RT-PCR values for GFP were normalized to 18S rRNA values. Error bars were derived from variation in values obtained from technical replicates performed in triplicate. (B) 293 cells were co-transfected with GFP and vector, ZEBRA, Z(S186A), Z(S186E), or Z(N182K). Cell extracts were prepared 45 h after transfection and analyzed by SDS-page. Immunoblots were probed with antibody specific for GFP, ZEBRA, and β-actin. The levels of GFP were quantified by densitometry and normalized to levels of β-actin.

    Techniques Used: Mutagenesis, Expressing, Transfection, Quantitative RT-PCR, Derivative Assay, Plasmid Preparation, SDS Page, Western Blot

    Frequency and intensity of PABPC-translocation induced by ZEBRA and BGLF5. 293 cells were transfected with vector, ZEBRA, or EGFP-BGLF5, or co-transfected with ZEBRA and EGFP-BGLF5. Cells were fixed and stained with antibodies specific for PABPC and ZEBRA, and fluorophore-conjugated secondary antibodies. Digital images were acquired by confocal microscopy and analyzed by ImageJ software (NIH). (A) Numbers of cells that were positive and negative for translocation of PABPC for each transfection condition. (B) Concentrations of intranuclear PABPC were measured by ImageJ software; 34 to 47 cells selected at random for each transfection condition. Measurements of intranuclear PABPC were normalized to the mean average value of 1.00 for the empty vector control.
    Figure Legend Snippet: Frequency and intensity of PABPC-translocation induced by ZEBRA and BGLF5. 293 cells were transfected with vector, ZEBRA, or EGFP-BGLF5, or co-transfected with ZEBRA and EGFP-BGLF5. Cells were fixed and stained with antibodies specific for PABPC and ZEBRA, and fluorophore-conjugated secondary antibodies. Digital images were acquired by confocal microscopy and analyzed by ImageJ software (NIH). (A) Numbers of cells that were positive and negative for translocation of PABPC for each transfection condition. (B) Concentrations of intranuclear PABPC were measured by ImageJ software; 34 to 47 cells selected at random for each transfection condition. Measurements of intranuclear PABPC were normalized to the mean average value of 1.00 for the empty vector control.

    Techniques Used: Translocation Assay, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Software

    39) Product Images from "Tools for Gene-Regulatory Analyses in the Marine Annelid Platynereis dumerilii"

    Article Title: Tools for Gene-Regulatory Analyses in the Marine Annelid Platynereis dumerilii

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093076

    Tol2-mediated transgenesis with pTol2{rps9::egfp} yields robust, ubiquitous expression of EGFP in G 0 animals. ( A, B ) pTol2{rps9::egfp} frkt868 -injected animals co-injected with tol2 mRNA (A) show significantly stronger EGFP fluorescence (green arrows) than those without transposase (B) (ae: autofluorescence around the adult eyes). ( C, D ) Stable ubiquitous EGFP expression (green arrows) two months after co-injection of donor DNA and tol2 mRNA (C) compared to a non-injected animal (D). Labels indicate autofluorescence of the jaws (j) as well as the iridophore pigments (p) in both the head and trunk.
    Figure Legend Snippet: Tol2-mediated transgenesis with pTol2{rps9::egfp} yields robust, ubiquitous expression of EGFP in G 0 animals. ( A, B ) pTol2{rps9::egfp} frkt868 -injected animals co-injected with tol2 mRNA (A) show significantly stronger EGFP fluorescence (green arrows) than those without transposase (B) (ae: autofluorescence around the adult eyes). ( C, D ) Stable ubiquitous EGFP expression (green arrows) two months after co-injection of donor DNA and tol2 mRNA (C) compared to a non-injected animal (D). Labels indicate autofluorescence of the jaws (j) as well as the iridophore pigments (p) in both the head and trunk.

    Techniques Used: Expressing, Injection, Fluorescence

    Excision of a microinjected Tol2-based construct. ( A ) Scheme of donor vector pTol2{rps9::egfp} frkt868 ; the rps::egfp cassette is flanked by inverted Tol2 repeats (Tol2IR) that serve as recognition site for Tol2 transposase co-injected as mRNA along with the vector DNA. ( B, C ) PCR using vector-specific primers (black arrows) yields a 200 bp PCR fragment specifically from embryos co-injected with tol2 transposase mRNA (left lane), but not from controls (right lane). ( D ) Precise cleavage of the reporter construct at the end of the Tol2 IRs is evidenced by sequencing of the 200 bp fragment.
    Figure Legend Snippet: Excision of a microinjected Tol2-based construct. ( A ) Scheme of donor vector pTol2{rps9::egfp} frkt868 ; the rps::egfp cassette is flanked by inverted Tol2 repeats (Tol2IR) that serve as recognition site for Tol2 transposase co-injected as mRNA along with the vector DNA. ( B, C ) PCR using vector-specific primers (black arrows) yields a 200 bp PCR fragment specifically from embryos co-injected with tol2 transposase mRNA (left lane), but not from controls (right lane). ( D ) Precise cleavage of the reporter construct at the end of the Tol2 IRs is evidenced by sequencing of the 200 bp fragment.

    Techniques Used: Construct, Plasmid Preparation, Injection, Polymerase Chain Reaction, Sequencing

    Excision of a microinjected Mos-based construct. ( A ) Scheme of donor vector pMos{rps9::egfp} frkt1074 ; in analogy to pTol2{rps9::egfp} frkt868 ( Figure 1A ), the rps::egfp cassette is flanked by inverted Mos repeats (MosIR) that serve as recognition site for Mos1 transposase. ( B, C ) PCR using vector-specific primers (black arrows) yields a variety PCR fragment from embryos co-injected with mos1 transposase mRNA (left lanes), but not controls (right lanes). ( D ) Sequencing of 21 individual PCR product reveals imprecise breakpoints caused by impaired excision of reporter constructs and/or subsequent modification of DNA ends in the context of non-homologous end repair. See Alignments S1 and S2 for details.
    Figure Legend Snippet: Excision of a microinjected Mos-based construct. ( A ) Scheme of donor vector pMos{rps9::egfp} frkt1074 ; in analogy to pTol2{rps9::egfp} frkt868 ( Figure 1A ), the rps::egfp cassette is flanked by inverted Mos repeats (MosIR) that serve as recognition site for Mos1 transposase. ( B, C ) PCR using vector-specific primers (black arrows) yields a variety PCR fragment from embryos co-injected with mos1 transposase mRNA (left lanes), but not controls (right lanes). ( D ) Sequencing of 21 individual PCR product reveals imprecise breakpoints caused by impaired excision of reporter constructs and/or subsequent modification of DNA ends in the context of non-homologous end repair. See Alignments S1 and S2 for details.

    Techniques Used: Construct, Plasmid Preparation, Polymerase Chain Reaction, Injection, Sequencing, Modification

    40) Product Images from "Neprilysin Is Poorly Expressed in the Prefrontal Cortex of Aged Dogs with Cognitive Dysfunction Syndrome"

    Article Title: Neprilysin Is Poorly Expressed in the Prefrontal Cortex of Aged Dogs with Cognitive Dysfunction Syndrome

    Journal: International Journal of Alzheimer's Disease

    doi: 10.1155/2014/483281

    Agarose gel electrophoresis of RT-PCR of NEP, IDE, and Ubi arranged by groups ((a) young, (b) aged-CU and (c) aged-CI). Amplicons of NEP cDNA (625 bp band) in young (a) and aged-CU (b) animals showed considerable intragroup variability; in contrast in aged-CI dogs (c), there are lower levels of NEP with less variability. IDE amplicons (493 bp band) present high intensity of band with similar levels in all animals. Ubi was included as an internal standard.
    Figure Legend Snippet: Agarose gel electrophoresis of RT-PCR of NEP, IDE, and Ubi arranged by groups ((a) young, (b) aged-CU and (c) aged-CI). Amplicons of NEP cDNA (625 bp band) in young (a) and aged-CU (b) animals showed considerable intragroup variability; in contrast in aged-CI dogs (c), there are lower levels of NEP with less variability. IDE amplicons (493 bp band) present high intensity of band with similar levels in all animals. Ubi was included as an internal standard.

    Techniques Used: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    41) Product Images from "Identification and characterization of the Plasmodium vivax thrombospondin-related apical merozoite protein"

    Article Title: Identification and characterization of the Plasmodium vivax thrombospondin-related apical merozoite protein

    Journal: Malaria Journal

    doi: 10.1186/1475-2875-9-283

    (A) Schematic representation of PvTRAMP indicating the localizations of the predicted signal peptide and transmembrane domain (both in dark gray), as well as the TSR domain (light gray) . Localization of the conserved cysteines inside the TSR domain and the synthetic peptides used in this study to obtain anti-PvTRAMP antisera are indicated by arrow heads and white boxes, respectively. (B) PCR amplification of pvtramp from P. vivax genomic DNA and cDNA. Lane 1. Amplification from genomic DNA using primers designed based on the sequence predicted for pvtramp . Lane 2. RT-PCR product amplified from DNAse-treated total P. vivax RNA. (C) Recognition of purified rPvTRAMP by anti-PvTRAMP rabbit sera, as assessed by Western blot. Lane 1: pre-immune sera. Lane 2: hyperimmune sera. Lane H: recognition of purified rPvTRAMP by anti-polyhistidine monoclonal antibody. (D) Western blot analysis of a P. vivax lysate with anti-PvTRAMP rabbit sera. Lane 1: pre-immune sera. Lane 2: hyperimmune sera.
    Figure Legend Snippet: (A) Schematic representation of PvTRAMP indicating the localizations of the predicted signal peptide and transmembrane domain (both in dark gray), as well as the TSR domain (light gray) . Localization of the conserved cysteines inside the TSR domain and the synthetic peptides used in this study to obtain anti-PvTRAMP antisera are indicated by arrow heads and white boxes, respectively. (B) PCR amplification of pvtramp from P. vivax genomic DNA and cDNA. Lane 1. Amplification from genomic DNA using primers designed based on the sequence predicted for pvtramp . Lane 2. RT-PCR product amplified from DNAse-treated total P. vivax RNA. (C) Recognition of purified rPvTRAMP by anti-PvTRAMP rabbit sera, as assessed by Western blot. Lane 1: pre-immune sera. Lane 2: hyperimmune sera. Lane H: recognition of purified rPvTRAMP by anti-polyhistidine monoclonal antibody. (D) Western blot analysis of a P. vivax lysate with anti-PvTRAMP rabbit sera. Lane 1: pre-immune sera. Lane 2: hyperimmune sera.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Reverse Transcription Polymerase Chain Reaction, Purification, Western Blot

    42) Product Images from "Identification and characterization of CRT10 as a novel regulator of Saccharomyces cerevisiae ribonucleotide reductase genes"

    Article Title: Identification and characterization of CRT10 as a novel regulator of Saccharomyces cerevisiae ribonucleotide reductase genes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl100

    ( A ) CRT10 expression in response to DNA damage and HU treatment. Log-phase wild-type HK578-10A cells were either untreated (lane 1) or treated with 0.3% MMS for 2 h (lane 2), 0.2 M HU for 2 h (lane 3) or exposed to 40 krad of γ radiation (lane 4). ( B ) CRT10 induction is DUN1 -dependent. Log-phase wild-type BY4741 and its derivatives WXY1153 ( crt1 Δ) and WXY1155 ( dun1 Δ) were either untreated (−) or treated with 0.1% MMS for 2 h (+). Northern hybridization was performed as described in Materials and Methods. The membranes were hybridized with CRT10 (upper panel), stripped and then hybridized with ACT1 (lower panel) as an internal control. Each lane contains 15 μg of total RNA.
    Figure Legend Snippet: ( A ) CRT10 expression in response to DNA damage and HU treatment. Log-phase wild-type HK578-10A cells were either untreated (lane 1) or treated with 0.3% MMS for 2 h (lane 2), 0.2 M HU for 2 h (lane 3) or exposed to 40 krad of γ radiation (lane 4). ( B ) CRT10 induction is DUN1 -dependent. Log-phase wild-type BY4741 and its derivatives WXY1153 ( crt1 Δ) and WXY1155 ( dun1 Δ) were either untreated (−) or treated with 0.1% MMS for 2 h (+). Northern hybridization was performed as described in Materials and Methods. The membranes were hybridized with CRT10 (upper panel), stripped and then hybridized with ACT1 (lower panel) as an internal control. Each lane contains 15 μg of total RNA.

    Techniques Used: Expressing, Northern Blot, Hybridization

    43) Product Images from "Tumor cell invasion of von Hippel Lindau renal cell carcinoma cells is mediated by membrane type-1 matrix metalloproteinase"

    Article Title: Tumor cell invasion of von Hippel Lindau renal cell carcinoma cells is mediated by membrane type-1 matrix metalloproteinase

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-5-66

    Expression of HIF-2α or MT1-MMP in WT8 cells increases matrix degradation and invasion . A . Collagen degradation. WT8 cells were transfected with either a control empty vector or pCMV-HIF-2α. Transfectants were serum-starved overnight, harvested and then embedded in a mixture of type I collagen and serum-free DMEM for the collagen degradation assay as in Fig 1. After 48 hours, the overlying media was weighed, and collagen degradation was determined by the volume of media liberated from the collagen gel. Values represent the average μL of media released from six samples (mean+/-S.D.); P
    Figure Legend Snippet: Expression of HIF-2α or MT1-MMP in WT8 cells increases matrix degradation and invasion . A . Collagen degradation. WT8 cells were transfected with either a control empty vector or pCMV-HIF-2α. Transfectants were serum-starved overnight, harvested and then embedded in a mixture of type I collagen and serum-free DMEM for the collagen degradation assay as in Fig 1. After 48 hours, the overlying media was weighed, and collagen degradation was determined by the volume of media liberated from the collagen gel. Values represent the average μL of media released from six samples (mean+/-S.D.); P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Degradation Assay

    Specific inhibition of MT1-MMP blocks HIF-2α mediated RCC tumor cell invasion . A . MT1-MMP protein expression as quantitated by an MT1-MMP ELISA activity assay. WT8 cells were transfected for 48 hours with pCMV-HIF-2α and a control or 3 specific MT1-MMP siRNA oligos. Each transfectant was additionally co-transfected with pCMV-eGFP. Transfection efficiency was consistent among the transfectants and was approximately 50%. Values represent the average [ng/mL] MT1-MMP of three transfections normalized to [μg/mL] total protein and are representative of three experiments (mean +/-S.D.); P
    Figure Legend Snippet: Specific inhibition of MT1-MMP blocks HIF-2α mediated RCC tumor cell invasion . A . MT1-MMP protein expression as quantitated by an MT1-MMP ELISA activity assay. WT8 cells were transfected for 48 hours with pCMV-HIF-2α and a control or 3 specific MT1-MMP siRNA oligos. Each transfectant was additionally co-transfected with pCMV-eGFP. Transfection efficiency was consistent among the transfectants and was approximately 50%. Values represent the average [ng/mL] MT1-MMP of three transfections normalized to [μg/mL] total protein and are representative of three experiments (mean +/-S.D.); P

    Techniques Used: Inhibition, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, Transfection

    44) Product Images from "Tumor cell invasion of von Hippel Lindau renal cell carcinoma cells is mediated by membrane type-1 matrix metalloproteinase"

    Article Title: Tumor cell invasion of von Hippel Lindau renal cell carcinoma cells is mediated by membrane type-1 matrix metalloproteinase

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-5-66

    Inhibition of MT1-MMP blocks RCC tumor cell invasion . (A) and (B) WT8 cells were transfected for 48 hours with MTpc3SE and control or 3 specific MT1-MMP siRNA oligos. Each transfectant was additionally co-transfected with pCMV-eGFP. Transfection efficiency was consistent among the transfectants and was approximately 50%. A . MT1-MMP protein expression in the transfectants as quantitated by an MT1-MMP ELISA activity assay. Values represent the average [ng/mL] MT1-MMP of three transfections normalized to [μg/mL] total protein and are representative of three experiments (mean+/-S.D.); P
    Figure Legend Snippet: Inhibition of MT1-MMP blocks RCC tumor cell invasion . (A) and (B) WT8 cells were transfected for 48 hours with MTpc3SE and control or 3 specific MT1-MMP siRNA oligos. Each transfectant was additionally co-transfected with pCMV-eGFP. Transfection efficiency was consistent among the transfectants and was approximately 50%. A . MT1-MMP protein expression in the transfectants as quantitated by an MT1-MMP ELISA activity assay. Values represent the average [ng/mL] MT1-MMP of three transfections normalized to [μg/mL] total protein and are representative of three experiments (mean+/-S.D.); P

    Techniques Used: Inhibition, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay

    Specific inhibition of MT1-MMP blocks HIF-2α mediated RCC tumor cell invasion . A . MT1-MMP protein expression as quantitated by an MT1-MMP ELISA activity assay. WT8 cells were transfected for 48 hours with pCMV-HIF-2α and a control or 3 specific MT1-MMP siRNA oligos. Each transfectant was additionally co-transfected with pCMV-eGFP. Transfection efficiency was consistent among the transfectants and was approximately 50%. Values represent the average [ng/mL] MT1-MMP of three transfections normalized to [μg/mL] total protein and are representative of three experiments (mean +/-S.D.); P
    Figure Legend Snippet: Specific inhibition of MT1-MMP blocks HIF-2α mediated RCC tumor cell invasion . A . MT1-MMP protein expression as quantitated by an MT1-MMP ELISA activity assay. WT8 cells were transfected for 48 hours with pCMV-HIF-2α and a control or 3 specific MT1-MMP siRNA oligos. Each transfectant was additionally co-transfected with pCMV-eGFP. Transfection efficiency was consistent among the transfectants and was approximately 50%. Values represent the average [ng/mL] MT1-MMP of three transfections normalized to [μg/mL] total protein and are representative of three experiments (mean +/-S.D.); P

    Techniques Used: Inhibition, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, Transfection

    45) Product Images from "Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line"

    Article Title: Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094401

    CHIKV structural polyprotein expression and processing in AcMNPV-CHIKV37997 infected Sf 21 cells and pV1JNS-CHIKV37997 transfected HEK293 cells. Cell lysate Western blots depicting ( A ) E2 expression and processing, detected by an E2 peptide-specific antibody. ( B ) E1 expression, detected by an E1 peptide-specific antibody. ( C ) E1/E2 (co-migrating) and capsid expression and processing, detected by an anti-CHIKV polyclonal antibody. AcMNPV-GFP infected Sf 21 lysate was included as a negative control for insect cells and baculovirus vector.
    Figure Legend Snippet: CHIKV structural polyprotein expression and processing in AcMNPV-CHIKV37997 infected Sf 21 cells and pV1JNS-CHIKV37997 transfected HEK293 cells. Cell lysate Western blots depicting ( A ) E2 expression and processing, detected by an E2 peptide-specific antibody. ( B ) E1 expression, detected by an E1 peptide-specific antibody. ( C ) E1/E2 (co-migrating) and capsid expression and processing, detected by an anti-CHIKV polyclonal antibody. AcMNPV-GFP infected Sf 21 lysate was included as a negative control for insect cells and baculovirus vector.

    Techniques Used: Expressing, Infection, Transfection, Western Blot, Negative Control, Plasmid Preparation

    Effect of elevated culture pH on cell-surface localization of CHIKV glycoproteins and CHIKV VLP yield. ( A ) Mean fluorescence intensity (MFI) from surface staining of AcMNPV-CHIKV37997 infected Sf 21 and pV1JNS-CHIKV37997 transfected HEK293 cells with neutralizing antibody m242. AcMNPV-NC and a mock transfection were included as negative controls for Sf 21 and HEK293, respectively. Error bars represent 95% confidence intervals (N = 3 independent infections/transfections). ( B ) E1/E2 Western blot and VLP concentration for supernatants from infected Sf 21 and transfected HEK293. Error bars represent 95% confidence intervals (N = 4 assay replicates).
    Figure Legend Snippet: Effect of elevated culture pH on cell-surface localization of CHIKV glycoproteins and CHIKV VLP yield. ( A ) Mean fluorescence intensity (MFI) from surface staining of AcMNPV-CHIKV37997 infected Sf 21 and pV1JNS-CHIKV37997 transfected HEK293 cells with neutralizing antibody m242. AcMNPV-NC and a mock transfection were included as negative controls for Sf 21 and HEK293, respectively. Error bars represent 95% confidence intervals (N = 3 independent infections/transfections). ( B ) E1/E2 Western blot and VLP concentration for supernatants from infected Sf 21 and transfected HEK293. Error bars represent 95% confidence intervals (N = 4 assay replicates).

    Techniques Used: Fluorescence, Staining, Infection, Transfection, Western Blot, Concentration Assay

    Biophysical characterization of CHIKV VLPs derived from SfBasic cells and comparison to a VLP standard derived from HEK293 cells. ( A ) Western blot of density gradient ultracentrifugation fractions containing CHIKV VLPs, using E1, E2, and capsid peptide-specific antibodies. ( B ) Dynamic light scattering (DLS) distribution of purified CHIKV VLP diameters (Ø). ( C ) Raw/unprocessed and 2D class average transmission electron microscopy images of purified CHIKV VLPs.
    Figure Legend Snippet: Biophysical characterization of CHIKV VLPs derived from SfBasic cells and comparison to a VLP standard derived from HEK293 cells. ( A ) Western blot of density gradient ultracentrifugation fractions containing CHIKV VLPs, using E1, E2, and capsid peptide-specific antibodies. ( B ) Dynamic light scattering (DLS) distribution of purified CHIKV VLP diameters (Ø). ( C ) Raw/unprocessed and 2D class average transmission electron microscopy images of purified CHIKV VLPs.

    Techniques Used: Derivative Assay, Western Blot, Purification, Transmission Assay, Electron Microscopy

    Immunogenicity assay titers from guinea pig sera after vaccination with adjuvanted Sf Basic-derived and HEK293-derived CHIKV VLPs. ( A ) Anti-CHIKV IgG geomean titer and CHIKV 181/25 geomean neutralizing titer (NT100) at study day 14 (14 days after first dose). ( B ) Anti-CHIKV IgG geomean titer and CHIKV 181/25 geomean neutralizing titer (NT100) at study day 21 (7 days after second dose). Geomean IgG ELISA background from pre-vaccination sera is indicated by a dashed line. Error bars represent 95% confidence intervals (N = 4 animals per group), and asterisks indicate a statistically significant increase in titer over background (p
    Figure Legend Snippet: Immunogenicity assay titers from guinea pig sera after vaccination with adjuvanted Sf Basic-derived and HEK293-derived CHIKV VLPs. ( A ) Anti-CHIKV IgG geomean titer and CHIKV 181/25 geomean neutralizing titer (NT100) at study day 14 (14 days after first dose). ( B ) Anti-CHIKV IgG geomean titer and CHIKV 181/25 geomean neutralizing titer (NT100) at study day 21 (7 days after second dose). Geomean IgG ELISA background from pre-vaccination sera is indicated by a dashed line. Error bars represent 95% confidence intervals (N = 4 animals per group), and asterisks indicate a statistically significant increase in titer over background (p

    Techniques Used: Immunogenicity Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay

    Transmission electron microscopy (TEM) images of thin-sections of AcMNPV-CHIKV37997 infected Sf 21 cells and pV1JNS-CHIKV37997 transfected HEK293 cells. ( A ) Putative CHIKV capsids formed in the cytoplasm of HEK293 cells. Capsid diameters are approximately 30–35 nm. ( B ) Baculovirus showing hallmark multiple nucleocapsids per envelope, with very electron dense nucleocapsids. Nucleocapsid diameter is approximately 40 nm. ( C ) Putative CHIKV capsids formed in the cytoplasm of Sf 21 cells. Capsid diameters are approximately 30–35 nm. Scale bar is equivalent for all images and represents 500 nm.
    Figure Legend Snippet: Transmission electron microscopy (TEM) images of thin-sections of AcMNPV-CHIKV37997 infected Sf 21 cells and pV1JNS-CHIKV37997 transfected HEK293 cells. ( A ) Putative CHIKV capsids formed in the cytoplasm of HEK293 cells. Capsid diameters are approximately 30–35 nm. ( B ) Baculovirus showing hallmark multiple nucleocapsids per envelope, with very electron dense nucleocapsids. Nucleocapsid diameter is approximately 40 nm. ( C ) Putative CHIKV capsids formed in the cytoplasm of Sf 21 cells. Capsid diameters are approximately 30–35 nm. Scale bar is equivalent for all images and represents 500 nm.

    Techniques Used: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Infection, Transfection

    46) Product Images from "Oral and Vaginal Epithelial Cell Lines Bind and Transfer Cell-Free Infectious HIV-1 to Permissive Cells but Are Not Productively Infected"

    Article Title: Oral and Vaginal Epithelial Cell Lines Bind and Transfer Cell-Free Infectious HIV-1 to Permissive Cells but Are Not Productively Infected

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098077

    Different methods used to detect HIV-1 R5 and X4 binding to epithelial cells. (A) Post-lysis detection of p24 gag protein by Western blotting. Primary (gingival) epithelial cells, TR146, FaDu, A431 and TZM-bl cells were incubated overnight (16–24 h) with cell free YU2 (R5) or LAI (X4). After extensive washing to remove unbound virus, normalised total protein lysates were separated by SDS-PAGE and probed for HIV p24 using α-actin as a loading control. (B) Detection of immobilized virus on the cell surface by flow cytometry. Epithelial cells were incubated overnight with cell free virus. Bound virus was detected using a Cy5-labeled anti-human secondary antibody to detect HIV-1 gp120 primary monoclonal on the APC channel. Electronic gates were set around an unlabelled cell control, this area is then set as zero and any cells shifted to the right of the gate are deemed positive. To determine amount of virus bound, virally exposed, labelled cell percentages are subtracted from the uninfected (unexposed) labelled control cell percentages to obtain the % fluorescence values shown. Data are representative of four independent experiments and bars indicate ± standard deviation from the mean. (C) Detection of packaged HIV R5 RNA by amplification of the HIV-1 pol gene using nested PCR. Total RNA was extracted from TR146, FaDu, A431 and TZM-bl cells incubated overnight with cell free YU2 (R5) and used to produce viral cDNA. This was then used as a template in a nested PCR to detect a 2 Kb region of HIV pol. (D) Percentage reduction in detection of immobilized virus on the cell surface by flow cytometry after trypsin treatment. Virally exposed cells are compared with cells labelled with secondary antibody alone. Data set is representative of three independent experiments. * = P
    Figure Legend Snippet: Different methods used to detect HIV-1 R5 and X4 binding to epithelial cells. (A) Post-lysis detection of p24 gag protein by Western blotting. Primary (gingival) epithelial cells, TR146, FaDu, A431 and TZM-bl cells were incubated overnight (16–24 h) with cell free YU2 (R5) or LAI (X4). After extensive washing to remove unbound virus, normalised total protein lysates were separated by SDS-PAGE and probed for HIV p24 using α-actin as a loading control. (B) Detection of immobilized virus on the cell surface by flow cytometry. Epithelial cells were incubated overnight with cell free virus. Bound virus was detected using a Cy5-labeled anti-human secondary antibody to detect HIV-1 gp120 primary monoclonal on the APC channel. Electronic gates were set around an unlabelled cell control, this area is then set as zero and any cells shifted to the right of the gate are deemed positive. To determine amount of virus bound, virally exposed, labelled cell percentages are subtracted from the uninfected (unexposed) labelled control cell percentages to obtain the % fluorescence values shown. Data are representative of four independent experiments and bars indicate ± standard deviation from the mean. (C) Detection of packaged HIV R5 RNA by amplification of the HIV-1 pol gene using nested PCR. Total RNA was extracted from TR146, FaDu, A431 and TZM-bl cells incubated overnight with cell free YU2 (R5) and used to produce viral cDNA. This was then used as a template in a nested PCR to detect a 2 Kb region of HIV pol. (D) Percentage reduction in detection of immobilized virus on the cell surface by flow cytometry after trypsin treatment. Virally exposed cells are compared with cells labelled with secondary antibody alone. Data set is representative of three independent experiments. * = P

    Techniques Used: Binding Assay, Lysis, Western Blot, Incubation, SDS Page, Flow Cytometry, Cytometry, Labeling, Fluorescence, Standard Deviation, Amplification, Nested PCR

    Post-integration HIV-1 mRNA transcription and de novo viral protein production in epithelial cells (MOI: 0.2). (A) Detection of spliced HIV-1 tat mRNA in TR146, FaDu, A431 and TZM-bl control cells by PCR 24 h post-infection with YU2 (R5) or LAI (X4) infectious virus. Equal amounts of total RNA was used to synthesise viral cDNA which was then subjected to PCR using primers designed to span the TAT 1 and 2 exon junctions. (B) p55 gag protein detection in TR146, FaDu, A431 and TZM-bl control cells by Western blot after 24 h infection with R5 (YU2) and LAI (X4) virus. (C) Infection of TR146, FaDu, A431 and NP2-R5/X4 control cells with GFP-linked single-cycle X4, R5 and dual tropic HIV-1 gp160 pseudotyped virus and detection of GFP incorporation into epithelial cell DNA by flow cytometry. Error bars show standard error from the mean. Data are representative of three independent experiments.
    Figure Legend Snippet: Post-integration HIV-1 mRNA transcription and de novo viral protein production in epithelial cells (MOI: 0.2). (A) Detection of spliced HIV-1 tat mRNA in TR146, FaDu, A431 and TZM-bl control cells by PCR 24 h post-infection with YU2 (R5) or LAI (X4) infectious virus. Equal amounts of total RNA was used to synthesise viral cDNA which was then subjected to PCR using primers designed to span the TAT 1 and 2 exon junctions. (B) p55 gag protein detection in TR146, FaDu, A431 and TZM-bl control cells by Western blot after 24 h infection with R5 (YU2) and LAI (X4) virus. (C) Infection of TR146, FaDu, A431 and NP2-R5/X4 control cells with GFP-linked single-cycle X4, R5 and dual tropic HIV-1 gp160 pseudotyped virus and detection of GFP incorporation into epithelial cell DNA by flow cytometry. Error bars show standard error from the mean. Data are representative of three independent experiments.

    Techniques Used: Polymerase Chain Reaction, Infection, Western Blot, Flow Cytometry, Cytometry

    47) Product Images from "Eutrophication has no short-term effect on the Cymbastela stipitata holobiont"

    Article Title: Eutrophication has no short-term effect on the Cymbastela stipitata holobiont

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2014.00216

    Relative abundance of each bacterial phyla, plus class for Proteobacteria , within each treatment for (A) DNA-derived and (B) RNA-derived sequences . The three replicate individuals per treatment were averaged. For clarity, only the 50 most abundant operational taxonomic units (OTUs) were used to create the graphs.
    Figure Legend Snippet: Relative abundance of each bacterial phyla, plus class for Proteobacteria , within each treatment for (A) DNA-derived and (B) RNA-derived sequences . The three replicate individuals per treatment were averaged. For clarity, only the 50 most abundant operational taxonomic units (OTUs) were used to create the graphs.

    Techniques Used: Derivative Assay

    48) Product Images from "Glucocorticoid-Induced Reversal of Interleukin-1?-Stimulated Inflammatory Gene Expression in Human Oviductal Cells"

    Article Title: Glucocorticoid-Induced Reversal of Interleukin-1?-Stimulated Inflammatory Gene Expression in Human Oviductal Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097997

    Increased PTGS2 levels in OE-E6/E7 cells by IL1β and TNFα treatment is blocked by DEX. A, Cells were treated with 50/ml IL1β for 24 h or 48 h and Western blot analysis was performed for PTSG2 and tubulin. B, Cells were treated with 10 nM DEX or vehicle 30 h prior to treatment with IL1β or vehicle and harvested 24 h later. Western blot analysis was performed for PTGS2 and tubulin. C, Western blot analysis was performed on OE-E6/E7 cells for glucocorticoid receptor and tubulin levels with BT20 breast cancer cells used as positive control. D, Cells were treated with DEX or vehicle 30 h prior to treatment with TNFα or vehicle. Cells treated with TNFα alone were harvested at 24 and 48 h after treatment and cells treated with DEX+TNFα were harvested at the 48 h time point. Western blot analysis was performed for PTGS2 and tubulin. Histograms summarize quantification of PTGS2 levels normalized to tubulin in 3 to 6 immunoblots. Bars represent the mean ± SEM relative to control. Bars with different letters are statistically different from one another as determined by ANOVA followed by a Student-Newman-Keuls post-hoc multiple comparison test ( p
    Figure Legend Snippet: Increased PTGS2 levels in OE-E6/E7 cells by IL1β and TNFα treatment is blocked by DEX. A, Cells were treated with 50/ml IL1β for 24 h or 48 h and Western blot analysis was performed for PTSG2 and tubulin. B, Cells were treated with 10 nM DEX or vehicle 30 h prior to treatment with IL1β or vehicle and harvested 24 h later. Western blot analysis was performed for PTGS2 and tubulin. C, Western blot analysis was performed on OE-E6/E7 cells for glucocorticoid receptor and tubulin levels with BT20 breast cancer cells used as positive control. D, Cells were treated with DEX or vehicle 30 h prior to treatment with TNFα or vehicle. Cells treated with TNFα alone were harvested at 24 and 48 h after treatment and cells treated with DEX+TNFα were harvested at the 48 h time point. Western blot analysis was performed for PTGS2 and tubulin. Histograms summarize quantification of PTGS2 levels normalized to tubulin in 3 to 6 immunoblots. Bars represent the mean ± SEM relative to control. Bars with different letters are statistically different from one another as determined by ANOVA followed by a Student-Newman-Keuls post-hoc multiple comparison test ( p

    Techniques Used: Western Blot, Positive Control

    Transcript levels of IL8 , IL23A , PI3 and TACC2 following IL1β and/or DEX treatment. OE-E6/E7 cells were treated with 10 nM DEX or vehicle 30 h prior to treatment with 50 ng/ml IL1β or vehicle and harvested 18 h later. Total RNA was extracted and RT-qPCR was performed for IL8 (A), IL23A (B), PI3 (C), TACC2 (D) and were normalized to TBP . Bars represent the mean ± SEM (n = 3 biological replicates performed in triplicate). Bars with different letters are statistically different from one another as determined by ANOVA followed by a Student-Newman-Keuls post-hoc multiple comparison test ( p
    Figure Legend Snippet: Transcript levels of IL8 , IL23A , PI3 and TACC2 following IL1β and/or DEX treatment. OE-E6/E7 cells were treated with 10 nM DEX or vehicle 30 h prior to treatment with 50 ng/ml IL1β or vehicle and harvested 18 h later. Total RNA was extracted and RT-qPCR was performed for IL8 (A), IL23A (B), PI3 (C), TACC2 (D) and were normalized to TBP . Bars represent the mean ± SEM (n = 3 biological replicates performed in triplicate). Bars with different letters are statistically different from one another as determined by ANOVA followed by a Student-Newman-Keuls post-hoc multiple comparison test ( p

    Techniques Used: Quantitative RT-PCR

    Network analysis of differentially expressed genes in human FTE OE-E6/E7 cells treated with DEX. Module annotations were performed with false discovery rate (FDR)
    Figure Legend Snippet: Network analysis of differentially expressed genes in human FTE OE-E6/E7 cells treated with DEX. Module annotations were performed with false discovery rate (FDR)

    Techniques Used:

    49) Product Images from "Bovine viral diarrhea virus NS4B protein is an integral membrane protein associated with Golgi markers and rearranged host membranes"

    Article Title: Bovine viral diarrhea virus NS4B protein is an integral membrane protein associated with Golgi markers and rearranged host membranes

    Journal: Virology Journal

    doi: 10.1186/1743-422X-6-185

    A. Representative plaque assays of cytopathic (cp) BVDV in MDBK cells . Cells were infected with 10-fold serial dilutions of BVDV stocks from virus supernatant. After adsorption, monolayers were overlaid with DMEM/5% horse serum and 0.5% agarose plugs. After 72 h incubation, the plugs were removed and the monolayers stained with 1% crystal violet. B. Growth Kinetics of cp BVDV in MDBK cells. Cells were infected with BVDV at MOI of 0.1. The supernatant (media, diamonds) and cell lysates (lysate, squares) were harvested at the indicated time points. Viral titers were determined via plaque assay. The results are given as log10 pfu/ml. C. BVDV RNA synthesis at various times post infection. MDBK cells were infected with BVDV as above and total cellular RNA was collected at 0 h (after 1 h adsorption), 6 h, 12 h, 18 h, and 24 h.p.i. To determine the amount of viral RNA in the cells, RT-PCR was performed with a probe specific to BVDV NS4B sequence. The amount of BVDV RNA was determined relative to GAPDH. D. BVDV NS4B cDNA products from RT-PCR, prior to quantitation, were run on 0.8% agarose gel and stained with ethidium bromide. Notice the increase in cDNA product from 6 to 24 h post BVDV infection.
    Figure Legend Snippet: A. Representative plaque assays of cytopathic (cp) BVDV in MDBK cells . Cells were infected with 10-fold serial dilutions of BVDV stocks from virus supernatant. After adsorption, monolayers were overlaid with DMEM/5% horse serum and 0.5% agarose plugs. After 72 h incubation, the plugs were removed and the monolayers stained with 1% crystal violet. B. Growth Kinetics of cp BVDV in MDBK cells. Cells were infected with BVDV at MOI of 0.1. The supernatant (media, diamonds) and cell lysates (lysate, squares) were harvested at the indicated time points. Viral titers were determined via plaque assay. The results are given as log10 pfu/ml. C. BVDV RNA synthesis at various times post infection. MDBK cells were infected with BVDV as above and total cellular RNA was collected at 0 h (after 1 h adsorption), 6 h, 12 h, 18 h, and 24 h.p.i. To determine the amount of viral RNA in the cells, RT-PCR was performed with a probe specific to BVDV NS4B sequence. The amount of BVDV RNA was determined relative to GAPDH. D. BVDV NS4B cDNA products from RT-PCR, prior to quantitation, were run on 0.8% agarose gel and stained with ethidium bromide. Notice the increase in cDNA product from 6 to 24 h post BVDV infection.

    Techniques Used: Infection, Adsorption, Incubation, Staining, Plaque Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Quantitation Assay, Agarose Gel Electrophoresis

    50) Product Images from "Reactive Astrocytes in Glial Scar Attract Olfactory Ensheathing Cells Migration by Secreted TNF-? in Spinal Cord Lesion of Rat"

    Article Title: Reactive Astrocytes in Glial Scar Attract Olfactory Ensheathing Cells Migration by Secreted TNF-? in Spinal Cord Lesion of Rat

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008141

    TNFR1 is expressed on OECs. (A) The mRNA of TNFR1 (301 bp) but not TNFR2 (264 bp) was identified in OECs by RT-PCR. (B) Cultured OECs were double-stained with antibodies against P75 and TNFR1 after treatment with Triton X-100. Scale bar = 50 µm. (C) Western blotting analysis for the expression of TNFR1 cell lysates of cultured OECs. GAPDH blotting severed as the loading control.
    Figure Legend Snippet: TNFR1 is expressed on OECs. (A) The mRNA of TNFR1 (301 bp) but not TNFR2 (264 bp) was identified in OECs by RT-PCR. (B) Cultured OECs were double-stained with antibodies against P75 and TNFR1 after treatment with Triton X-100. Scale bar = 50 µm. (C) Western blotting analysis for the expression of TNFR1 cell lysates of cultured OECs. GAPDH blotting severed as the loading control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Staining, Western Blot, Expressing

    51) Product Images from "Cap-independent translation through the p27 5?-UTR"

    Article Title: Cap-independent translation through the p27 5?-UTR

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm512

    RT-PCR analysis of transcripts produced from bicistronic reporter plasmids. Top: Diagram of the transcripts that should be produced following transfection when transcription initiates from the TK promoter of the bicistronic reporter construct lacking an insert (pTKLL) and the construct carrying the p27 5′-UTR (pTKLL-472). Arrows below each diagram indicate the position of primers used for RT-PCR analysis. RT-PCR should produce a DNA fragment of ∼1635 nt if there is no splicing due to a cryptic splice acceptor site in the p27 5′-UTR. The expected size for the fragment from pTKLL transfected cells is ∼1160 nt. Bottom: The bicistronic reporters were transfected into MCF7 cells as indicated. The control reactions (C) were from untransfected cells. RNA was isolated and used for RT-PCR with the primer pair indicated in the diagram. Reactions in which all conditions were identical except that reverse transcriptase was omitted (−RT) were performed for each of the RNA templates.
    Figure Legend Snippet: RT-PCR analysis of transcripts produced from bicistronic reporter plasmids. Top: Diagram of the transcripts that should be produced following transfection when transcription initiates from the TK promoter of the bicistronic reporter construct lacking an insert (pTKLL) and the construct carrying the p27 5′-UTR (pTKLL-472). Arrows below each diagram indicate the position of primers used for RT-PCR analysis. RT-PCR should produce a DNA fragment of ∼1635 nt if there is no splicing due to a cryptic splice acceptor site in the p27 5′-UTR. The expected size for the fragment from pTKLL transfected cells is ∼1160 nt. Bottom: The bicistronic reporters were transfected into MCF7 cells as indicated. The control reactions (C) were from untransfected cells. RNA was isolated and used for RT-PCR with the primer pair indicated in the diagram. Reactions in which all conditions were identical except that reverse transcriptase was omitted (−RT) were performed for each of the RNA templates.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Produced, Transfection, Construct, Isolation

    52) Product Images from "Retroviral vectors for establishing tetracycline-regulated gene expression in an otherwise recalcitrant cell line"

    Article Title: Retroviral vectors for establishing tetracycline-regulated gene expression in an otherwise recalcitrant cell line

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-3-13

    Generation and analysis of modified RevTetOFF constructs. ( A ) Three additional constructs were generated in which the CMV promoter was replaced by either the elongation factor 1-alpha or phosphoglycerate kinase-1 promoters, or a tetracycline responsive element. ( B ) Parental HC11 cells were transiently co-transfected with the indicated constructs and maintained for 48 hours in the presence of absence of 1 μg/ml doxycycline. The cells were fixed and stained for β-galactosidase expression using X-gal 48 hours after transfection.
    Figure Legend Snippet: Generation and analysis of modified RevTetOFF constructs. ( A ) Three additional constructs were generated in which the CMV promoter was replaced by either the elongation factor 1-alpha or phosphoglycerate kinase-1 promoters, or a tetracycline responsive element. ( B ) Parental HC11 cells were transiently co-transfected with the indicated constructs and maintained for 48 hours in the presence of absence of 1 μg/ml doxycycline. The cells were fixed and stained for β-galactosidase expression using X-gal 48 hours after transfection.

    Techniques Used: Modification, Construct, Generated, Transfection, Staining, Expressing

    Lack of tTA expression prevents β-galactosidase induction in HC11(CMV- tTA /TRE- lacZ ) cells. The cells were transiently transfected with the indicated constructs and were cultured either in the presence ( A , C , E , G , H , I ) or absence ( B , D , F , H , J ) of 1 μg/ml doxycycline for 48 hours. The CMV- lacZ expression vector was used as a positive control for β-galactosidase expression. The cells were fixed and stained for β-galactosidase expression using X-gal 48 hours after transfection.
    Figure Legend Snippet: Lack of tTA expression prevents β-galactosidase induction in HC11(CMV- tTA /TRE- lacZ ) cells. The cells were transiently transfected with the indicated constructs and were cultured either in the presence ( A , C , E , G , H , I ) or absence ( B , D , F , H , J ) of 1 μg/ml doxycycline for 48 hours. The CMV- lacZ expression vector was used as a positive control for β-galactosidase expression. The cells were fixed and stained for β-galactosidase expression using X-gal 48 hours after transfection.

    Techniques Used: Expressing, Transfection, Construct, Cell Culture, Plasmid Preparation, Positive Control, Staining

    53) Product Images from "The Gs-Linked Receptor GPR3 Inhibits the Proliferation of Cerebellar Granule Cells during Postnatal Development"

    Article Title: The Gs-Linked Receptor GPR3 Inhibits the Proliferation of Cerebellar Granule Cells during Postnatal Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005922

    GPR3 expression during postnatal rat and mouse cerebellar development. Panel a: Total mRNA was extracted from rat cerebelli (N = 12–15 per time point) at 4 different developmental stages (P1, P7, P21) and adult (7 to 8 weeks). RNA samples were subjected to quantitative RT-PCR analysis using probes and primers specific to rat GPR3 . Data were adjusted using β-actin mRNA as control. Panel b: To determine the distribution of GPR3 in the developing postnatal cerebellum, a GPR3−/−; LacZ +/+ mouse was employed, where the E. coli LacZ gene was substituted into the GPR3 locus and was thus under transcriptional control of the endogenous GPR3 promoter. Staining for β-galactosidase expression revealed increased activation of transcriptional activity of the GPR3 promoter in the internal granular layer of cerebellum from P5 to P12 stages of postnatal development. Scale bar = 100 µm.
    Figure Legend Snippet: GPR3 expression during postnatal rat and mouse cerebellar development. Panel a: Total mRNA was extracted from rat cerebelli (N = 12–15 per time point) at 4 different developmental stages (P1, P7, P21) and adult (7 to 8 weeks). RNA samples were subjected to quantitative RT-PCR analysis using probes and primers specific to rat GPR3 . Data were adjusted using β-actin mRNA as control. Panel b: To determine the distribution of GPR3 in the developing postnatal cerebellum, a GPR3−/−; LacZ +/+ mouse was employed, where the E. coli LacZ gene was substituted into the GPR3 locus and was thus under transcriptional control of the endogenous GPR3 promoter. Staining for β-galactosidase expression revealed increased activation of transcriptional activity of the GPR3 promoter in the internal granular layer of cerebellum from P5 to P12 stages of postnatal development. Scale bar = 100 µm.

    Techniques Used: Expressing, Quantitative RT-PCR, Staining, Activation Assay, Activity Assay

    54) Product Images from "NEDDylation Is Essential for Kaposi’s Sarcoma-Associated Herpesvirus Latency and Lytic Reactivation and Represents a Novel Anti-KSHV Target"

    Article Title: NEDDylation Is Essential for Kaposi’s Sarcoma-Associated Herpesvirus Latency and Lytic Reactivation and Represents a Novel Anti-KSHV Target

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004771

    MLN4924 induced lytic cycle—associated expression. ( A ) Latently infected TREx-BCBL-1-RTA cells were treated with 1 μM MLN4924 and total RNA was isolated from samples harvested 24 h later. The normalized expression (using GAPDH expression) of lytic cycle genes and latency associated ( ORF73 , ORF71 and K12 ) genes were analyzed by qRT-PCR and compared to the levels found in DMSO-treated control samples. Three biological replicates were independently analyzed and the error bars represent the standard deviation from the mean. ( B ) The inhibition of NEDDylation leads to reactivation of lytic cycle protein expression; rKSHV.219 cells were treated with 0.1 μM MLN4924 (a concentration that was tolerated for the duration of the experiment) for 36 h. Red fluorescent protein (RFP) expression was observed in numerous MLN4924-treated cells, indicative of RTA protein expression. RFP expression was observed in a minority DMSO-only treated cells (as expected due to low level spontaneous reactivation). ( C ) ORF57 expression was also detected by immunoblot analysis of MLN4924-treated rKSHV.219 cells. ( D ) Inhibition of NEDDylation enhances reactivation. MLN4924-treated rKSHV.219 cells were incubated with or without TPA (able to reactivate KSHV) for 36 h.
    Figure Legend Snippet: MLN4924 induced lytic cycle—associated expression. ( A ) Latently infected TREx-BCBL-1-RTA cells were treated with 1 μM MLN4924 and total RNA was isolated from samples harvested 24 h later. The normalized expression (using GAPDH expression) of lytic cycle genes and latency associated ( ORF73 , ORF71 and K12 ) genes were analyzed by qRT-PCR and compared to the levels found in DMSO-treated control samples. Three biological replicates were independently analyzed and the error bars represent the standard deviation from the mean. ( B ) The inhibition of NEDDylation leads to reactivation of lytic cycle protein expression; rKSHV.219 cells were treated with 0.1 μM MLN4924 (a concentration that was tolerated for the duration of the experiment) for 36 h. Red fluorescent protein (RFP) expression was observed in numerous MLN4924-treated cells, indicative of RTA protein expression. RFP expression was observed in a minority DMSO-only treated cells (as expected due to low level spontaneous reactivation). ( C ) ORF57 expression was also detected by immunoblot analysis of MLN4924-treated rKSHV.219 cells. ( D ) Inhibition of NEDDylation enhances reactivation. MLN4924-treated rKSHV.219 cells were incubated with or without TPA (able to reactivate KSHV) for 36 h.

    Techniques Used: Expressing, Infection, Isolation, Quantitative RT-PCR, Standard Deviation, Inhibition, Concentration Assay, Incubation

    The role of individual CRLs for the regulation of KSHV latency. ( A ) Dominant-negative versions of each Cullin (FLAG-DNCul that inhibits CRL activity [ 35 ]) was expressed in latently infected rKSHV.219 cells and ORF57 expression (as a marker of lytic reactivation) was tested by immunoblot analysis 36 h later. Treatment of cells with MLN4924 and TPA/NaB (sodium butyrate) served as positive controls for reactivation. *denotes non-specific bands and error bars represent the standard deviation of the mean of two independent transfection experiments. ( B ) Densitometry analysis of (A). ( C ) qRT-PCR analysis of Cul4B mRNA from TREx-BCBL-1-RTA cells transfected with either a scramble control shRNA expression vector (Scr.) or four independent shRNA expression vectors targeted at Cul4B (shCul4B). RNA was harvested four days post transfection. ( D ) qRT-PCR analysis of viral gene expression in Cul4B-knockdown cells normalized against GAPDH expression. Error bars represent the standard deviation from three independent transfections per condition. OLT denotes OriLyt transcript.
    Figure Legend Snippet: The role of individual CRLs for the regulation of KSHV latency. ( A ) Dominant-negative versions of each Cullin (FLAG-DNCul that inhibits CRL activity [ 35 ]) was expressed in latently infected rKSHV.219 cells and ORF57 expression (as a marker of lytic reactivation) was tested by immunoblot analysis 36 h later. Treatment of cells with MLN4924 and TPA/NaB (sodium butyrate) served as positive controls for reactivation. *denotes non-specific bands and error bars represent the standard deviation of the mean of two independent transfection experiments. ( B ) Densitometry analysis of (A). ( C ) qRT-PCR analysis of Cul4B mRNA from TREx-BCBL-1-RTA cells transfected with either a scramble control shRNA expression vector (Scr.) or four independent shRNA expression vectors targeted at Cul4B (shCul4B). RNA was harvested four days post transfection. ( D ) qRT-PCR analysis of viral gene expression in Cul4B-knockdown cells normalized against GAPDH expression. Error bars represent the standard deviation from three independent transfections per condition. OLT denotes OriLyt transcript.

    Techniques Used: Dominant Negative Mutation, Activity Assay, Infection, Expressing, Marker, Standard Deviation, Transfection, Quantitative RT-PCR, shRNA, Plasmid Preparation

    55) Product Images from "Efficient CRISPR/Cas9-mediated genome editing in P. falciparum"

    Article Title: Efficient CRISPR/Cas9-mediated genome editing in P. falciparum

    Journal: Nature methods

    doi: 10.1038/nmeth.3063

    Validating T7 RNA polymerase functions in P. falciparum . (a) Schematic of the Cas9, sgRNA and target DNA interaction. The sgRNA base pairs with a 20 nucleotide target DNA region defined by an NGG protospacer adjacent motif (PAM). Cas9 induces double strand DNA cleavage (arrow heads) 3 nucleotides upstream of the PAM site. ( b ) Plasmids used to test T7 RNAP expression and activity. In the reporter plasmid, pT7 RL1, the Renilla luciferase ( RL ) gene is expressed from a T7 promoter- T7 terminator cassette. The pT7 RL2 control plasmid contains the RL gene and T7 terminator, but no T7 promoter. A second control plasmid, pΔT7, lacks the T7 promoter-RL gene- T7 terminator cassette. ( c ) Western blot analysis of T7 RNAP protein production in parasites. Blots were probed with anti-T7 RNAP and anti-NPTII (loading control) antibodies. ( d ) Quantitative RT-PCR analysis of normalized RL transcript levels produced after the indicated transfections. Levels are above background only for the pT7 RNAP plus pT7 RL1 case. Data shown are mean ± s.d. (n = 3 technical replicates). ( e ) Northern blot analysis for RL transcript production, with the bsd selection marker transcript probed as a control. ( f ) Relative parasite growth over four successive generations both in the presence or absence of T7 RNAP and a T7 promoter-driven expression cassette. Data are shown as mean ± s.d. (n = 3 biological replicates) and analyzed for significance by One-way ANOVA.
    Figure Legend Snippet: Validating T7 RNA polymerase functions in P. falciparum . (a) Schematic of the Cas9, sgRNA and target DNA interaction. The sgRNA base pairs with a 20 nucleotide target DNA region defined by an NGG protospacer adjacent motif (PAM). Cas9 induces double strand DNA cleavage (arrow heads) 3 nucleotides upstream of the PAM site. ( b ) Plasmids used to test T7 RNAP expression and activity. In the reporter plasmid, pT7 RL1, the Renilla luciferase ( RL ) gene is expressed from a T7 promoter- T7 terminator cassette. The pT7 RL2 control plasmid contains the RL gene and T7 terminator, but no T7 promoter. A second control plasmid, pΔT7, lacks the T7 promoter-RL gene- T7 terminator cassette. ( c ) Western blot analysis of T7 RNAP protein production in parasites. Blots were probed with anti-T7 RNAP and anti-NPTII (loading control) antibodies. ( d ) Quantitative RT-PCR analysis of normalized RL transcript levels produced after the indicated transfections. Levels are above background only for the pT7 RNAP plus pT7 RL1 case. Data shown are mean ± s.d. (n = 3 technical replicates). ( e ) Northern blot analysis for RL transcript production, with the bsd selection marker transcript probed as a control. ( f ) Relative parasite growth over four successive generations both in the presence or absence of T7 RNAP and a T7 promoter-driven expression cassette. Data are shown as mean ± s.d. (n = 3 biological replicates) and analyzed for significance by One-way ANOVA.

    Techniques Used: Expressing, Activity Assay, Plasmid Preparation, Luciferase, Western Blot, Quantitative RT-PCR, Produced, Transfection, Northern Blot, Selection, Marker

    56) Product Images from "The transcriptome of Utricularia vulgaris, a rootless plant with minimalist genome, reveals extreme alternative splicing and only moderate sequence similarity with Utricularia gibba"

    Article Title: The transcriptome of Utricularia vulgaris, a rootless plant with minimalist genome, reveals extreme alternative splicing and only moderate sequence similarity with Utricularia gibba

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-015-0467-8

    PCR amplification with the LCB-1-P lyase specific primers. An agarose gel (1.2%) electrophoresis of PCR fragments amplified from the gene encoding LCB-1-P lyase (isogroup 000007) in U. vulgaris with different templates. 1–6: cDNA prepared from RNA extracted from two different plant individuals, 7: genomic DNA. NC: negative control with water instead of DNA. (A) PCR with exon-specific primers UV405_F1 and UV405_R1. (B) . PCR with intron-specific primer UV405_F2 and exon-specific primer UV405_R1. Annealing temperature is indicated above the lanes. Standard of molecular weights is shown on the both sides of the gel.
    Figure Legend Snippet: PCR amplification with the LCB-1-P lyase specific primers. An agarose gel (1.2%) electrophoresis of PCR fragments amplified from the gene encoding LCB-1-P lyase (isogroup 000007) in U. vulgaris with different templates. 1–6: cDNA prepared from RNA extracted from two different plant individuals, 7: genomic DNA. NC: negative control with water instead of DNA. (A) PCR with exon-specific primers UV405_F1 and UV405_R1. (B) . PCR with intron-specific primer UV405_F2 and exon-specific primer UV405_R1. Annealing temperature is indicated above the lanes. Standard of molecular weights is shown on the both sides of the gel.

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Electrophoresis, Negative Control

    57) Product Images from "Single Molecule PCR Reveals Similar Patterns of Non-Homologous DSB Repair in Tobacco and Arabidopsis"

    Article Title: Single Molecule PCR Reveals Similar Patterns of Non-Homologous DSB Repair in Tobacco and Arabidopsis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032255

    Overview of EtOH induced induction of DSBs. The T-DNA of vector pAlcR:ISceI ( A ) contains the left (LB) and right border (RB) sequences; a hygromycin selectable marker gene ( hyg ); the AlcR gene constitutively expressed from the 35S promoter; and the I-SceI gene driven by the alcA :35S promoter. In the presence of EtOH, AlcR binds to and transcriptionally activates the alcA :35S promoter, driving expression of I-SceI . The T-DNA of vector pdao1 ( B ) contains left and right border sequences; a kanamycin selectable marker gene ( neo ); and a spacer region flanked by I-SceI target sites. The spacer region also contains three HincII sites (H). ( C ) Upon I-SceI expression the I-SceI sites are cleaved leading to the excision of the spacer region. DSB repair will then result in the joining of the cleaved sequences. This may result in direct joining of the I-SceI restriction sites, deletion of sequence on either side of the DSB or insertion of sequence at the site of DSB repair. These three types of repair can be distinguished by PCR using primers P1 (DSBF1) and P2 (DSBR1) that flank the site of DSB repair. Direct joining will result in an 834 bp product whilst deletion will result in a smaller product and insertion in a larger product. In the absence of DSB induction or when DSBs are repaired by HR the spacer region will not be excised resulting in a PCR product of 2.9 kb ( B ). Amplification of this product will be prevented by digesting template DNA with HincII.
    Figure Legend Snippet: Overview of EtOH induced induction of DSBs. The T-DNA of vector pAlcR:ISceI ( A ) contains the left (LB) and right border (RB) sequences; a hygromycin selectable marker gene ( hyg ); the AlcR gene constitutively expressed from the 35S promoter; and the I-SceI gene driven by the alcA :35S promoter. In the presence of EtOH, AlcR binds to and transcriptionally activates the alcA :35S promoter, driving expression of I-SceI . The T-DNA of vector pdao1 ( B ) contains left and right border sequences; a kanamycin selectable marker gene ( neo ); and a spacer region flanked by I-SceI target sites. The spacer region also contains three HincII sites (H). ( C ) Upon I-SceI expression the I-SceI sites are cleaved leading to the excision of the spacer region. DSB repair will then result in the joining of the cleaved sequences. This may result in direct joining of the I-SceI restriction sites, deletion of sequence on either side of the DSB or insertion of sequence at the site of DSB repair. These three types of repair can be distinguished by PCR using primers P1 (DSBF1) and P2 (DSBR1) that flank the site of DSB repair. Direct joining will result in an 834 bp product whilst deletion will result in a smaller product and insertion in a larger product. In the absence of DSB induction or when DSBs are repaired by HR the spacer region will not be excised resulting in a PCR product of 2.9 kb ( B ). Amplification of this product will be prevented by digesting template DNA with HincII.

    Techniques Used: Plasmid Preparation, Marker, Expressing, Sequencing, Polymerase Chain Reaction, Amplification

    PCR analysis of DSB induction and repair. ( A ) RT-PCR (+) demonstrates increased I-SceI mRNA accumulation after induction with 0.7 M ethanol in tobacco leaf tissue. Low levels of I-SceI mRNA accumulate in non-induced leaf tissue. No reverse transcriptase (−) and no template (-ve) controls are shown. Template control RT-PCRs used RPL25 mRNA primers. ( B ) The DSB region was PCR amplified from 4 tobacco D4A2 plants using primers DSBF1 and DSBR1 which flank the two I-SceI sites. Only the full length 2.9 kb band is amplified from template DNA extracted prior to DSB induction (NI). An additional ∼834 bp band is amplified from template DNA extracted after DSB induction (I). 834 bp is the expected size of the DSB region after excision of the spacer region. ( C ) After HincII digestion of induced template DNA only the 834 bp band is amplified. No amplification is observed from those molecules which have not undergone dao1 excision. Individual repair junctions were amplified by smPCR ( D – F ). The majority of products amplified were ∼834 bp in size. Some repair events resulted in deletions leading to products
    Figure Legend Snippet: PCR analysis of DSB induction and repair. ( A ) RT-PCR (+) demonstrates increased I-SceI mRNA accumulation after induction with 0.7 M ethanol in tobacco leaf tissue. Low levels of I-SceI mRNA accumulate in non-induced leaf tissue. No reverse transcriptase (−) and no template (-ve) controls are shown. Template control RT-PCRs used RPL25 mRNA primers. ( B ) The DSB region was PCR amplified from 4 tobacco D4A2 plants using primers DSBF1 and DSBR1 which flank the two I-SceI sites. Only the full length 2.9 kb band is amplified from template DNA extracted prior to DSB induction (NI). An additional ∼834 bp band is amplified from template DNA extracted after DSB induction (I). 834 bp is the expected size of the DSB region after excision of the spacer region. ( C ) After HincII digestion of induced template DNA only the 834 bp band is amplified. No amplification is observed from those molecules which have not undergone dao1 excision. Individual repair junctions were amplified by smPCR ( D – F ). The majority of products amplified were ∼834 bp in size. Some repair events resulted in deletions leading to products

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification

    58) Product Images from "Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection"

    Article Title: Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-12-83

    Genomic DNA PCR (A) and RT-PCR (B) analysis in Sf9/pIBdsCasp-1 and Sf9/pIBdsCasp-2 cells. Genomic DNA and total RNA of the cells (Sf9, Sf9/pIB, Sf9/pIBdsCasp-1 and Sf9/pIBdsCasp-2) were extracted and analyzed by PCR and RT-PCR, respectively. The corresponding specific primer sequences and PCR cycles are presented in Table 1 . pIB, Sf9/pIB cells; dsCasp-1, Sf9/pIBdsCasp-1 cells; dsCasp-2, Sf9/pIBdsCasp-2 cells.
    Figure Legend Snippet: Genomic DNA PCR (A) and RT-PCR (B) analysis in Sf9/pIBdsCasp-1 and Sf9/pIBdsCasp-2 cells. Genomic DNA and total RNA of the cells (Sf9, Sf9/pIB, Sf9/pIBdsCasp-1 and Sf9/pIBdsCasp-2) were extracted and analyzed by PCR and RT-PCR, respectively. The corresponding specific primer sequences and PCR cycles are presented in Table 1 . pIB, Sf9/pIB cells; dsCasp-1, Sf9/pIBdsCasp-1 cells; dsCasp-2, Sf9/pIBdsCasp-2 cells.

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    59) Product Images from "Cloning and expression of porcine Colony Stimulating Factor-1 (CSF-1) and Colony Stimulating Factor-1 Receptor (CSF-1R) and analysis of the species specificity of stimulation by CSF-1 and Interleukin 34"

    Article Title: Cloning and expression of porcine Colony Stimulating Factor-1 (CSF-1) and Colony Stimulating Factor-1 Receptor (CSF-1R) and analysis of the species specificity of stimulation by CSF-1 and Interleukin 34

    Journal: Cytokine

    doi: 10.1016/j.cyto.2012.08.008

    Expression of cloned porcine CSF-1 (A) and CSF-1R (B). (A) Western blot of secreted cloned porcine CSF-1 by HEK293T cells transfected with porcine CSF-1_pEF6 expression construct or empty pEF6 construct. Under non-reducing conditions, two bands of approximately 37 KDa and 50 KDa were detected, whereas two smaller bands (20 and 25 KDa) were detected in the presence of dithiothreitol (DTT). Porcine CSF-1 is secreted as a disulphide linked dimer that is variably glycosylated. (B) Western blot of cloned expressed porcine CSF-1R transfected into Ba/F3 cells. Cells were cultured in either rh-CSF-1, IL-3 or both factors combined prior to collection of cell lysate. Differential levels of receptor expression were noted when the cells were cultured in these different conditions. Receptor expression is reduced when Ba/F3 cells expressing CSF-1R are cultured in rh-CSF-1 alone.
    Figure Legend Snippet: Expression of cloned porcine CSF-1 (A) and CSF-1R (B). (A) Western blot of secreted cloned porcine CSF-1 by HEK293T cells transfected with porcine CSF-1_pEF6 expression construct or empty pEF6 construct. Under non-reducing conditions, two bands of approximately 37 KDa and 50 KDa were detected, whereas two smaller bands (20 and 25 KDa) were detected in the presence of dithiothreitol (DTT). Porcine CSF-1 is secreted as a disulphide linked dimer that is variably glycosylated. (B) Western blot of cloned expressed porcine CSF-1R transfected into Ba/F3 cells. Cells were cultured in either rh-CSF-1, IL-3 or both factors combined prior to collection of cell lysate. Differential levels of receptor expression were noted when the cells were cultured in these different conditions. Receptor expression is reduced when Ba/F3 cells expressing CSF-1R are cultured in rh-CSF-1 alone.

    Techniques Used: Expressing, Clone Assay, Western Blot, Transfection, Construct, Cell Culture

    Demonstration of biological activity of cloned secreted porcine CSF-1 transfected into HEK293T cells and porcine CSF-1 expressed in E.Coli . (A) An MTT cell viability assay was performed using Ba/F3 cells expressing porcine CSF-1R and supernatant collected from transfected HEK293T cells with porcine CSF-1_pEF6 expression construct. Using 80% of the HEK293T transfected cell supernatant produced viable cells. (B) Porcine bone marrow cells cultured with 20% supernatant collected from HEK293T cells transfected with porcine CSF-1_pEF6 expression construct differentiated into BMDMs, adhered to the tissue culture plate and proliferated compared to cells cultured with supernatant from HEK293T cells transfected with empty pEF6 construct which did not adhere, proliferate or survive after 7 days in culture (C). (D) Bacterially expressed Porcine CSF-1 has also shown to be biologically active on the porcine CSF-1R expressed in Ba/F3 cells in an MTT cell viability assay. (E) An MTT cell viability assay was performed using mouse BMMs cultured with supernatant collected from transfected HEK293T cells with porcine CSF-1_pEF6 expression construct. Using either 50% and 20% supernatant produced viable cells. All of the assays shown are representative of three separate experiments with either triplicate or quadruplicate determinations.
    Figure Legend Snippet: Demonstration of biological activity of cloned secreted porcine CSF-1 transfected into HEK293T cells and porcine CSF-1 expressed in E.Coli . (A) An MTT cell viability assay was performed using Ba/F3 cells expressing porcine CSF-1R and supernatant collected from transfected HEK293T cells with porcine CSF-1_pEF6 expression construct. Using 80% of the HEK293T transfected cell supernatant produced viable cells. (B) Porcine bone marrow cells cultured with 20% supernatant collected from HEK293T cells transfected with porcine CSF-1_pEF6 expression construct differentiated into BMDMs, adhered to the tissue culture plate and proliferated compared to cells cultured with supernatant from HEK293T cells transfected with empty pEF6 construct which did not adhere, proliferate or survive after 7 days in culture (C). (D) Bacterially expressed Porcine CSF-1 has also shown to be biologically active on the porcine CSF-1R expressed in Ba/F3 cells in an MTT cell viability assay. (E) An MTT cell viability assay was performed using mouse BMMs cultured with supernatant collected from transfected HEK293T cells with porcine CSF-1_pEF6 expression construct. Using either 50% and 20% supernatant produced viable cells. All of the assays shown are representative of three separate experiments with either triplicate or quadruplicate determinations.

    Techniques Used: Activity Assay, Clone Assay, Transfection, MTT Assay, Viability Assay, Expressing, Construct, Produced, Cell Culture

    60) Product Images from "Respiratory Syncytial Virus Fusion Glycoprotein Expressed in Insect Cells Form Protein Nanoparticles That Induce Protective Immunity in Cotton Rats"

    Article Title: Respiratory Syncytial Virus Fusion Glycoprotein Expressed in Insect Cells Form Protein Nanoparticles That Induce Protective Immunity in Cotton Rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0050852

    RSV A2 F gene modifications. (A) RSV F cleavage site mutation where introduced at the proteolytic furin cleavage site II (KKRKRR) and site I (RARR) of wild type RSV F gene (wt). (B) RSV F clone 2 (#514) with a mutation of cleavage site II was further modified with increasing deletions in the fusion peptide starting at Phe137 to Val154 with Δ2, Δ4, Δ6, Δ8, Δ10, Δ12, Δ14, Δ16 and Δ18 amino acid deletions. (C) The modified full length RSV F gene (#683) with a modified cleavage site II (KKQKQQ) and Phe137 - Ser146 fusion domain deletion including RSV F cleavable signal peptide (sp), 27 amino acid peptide [23] , hydrophobic transmembrane (tm), and F2 and F1 polypeptides covalently linked by two disulfides.
    Figure Legend Snippet: RSV A2 F gene modifications. (A) RSV F cleavage site mutation where introduced at the proteolytic furin cleavage site II (KKRKRR) and site I (RARR) of wild type RSV F gene (wt). (B) RSV F clone 2 (#514) with a mutation of cleavage site II was further modified with increasing deletions in the fusion peptide starting at Phe137 to Val154 with Δ2, Δ4, Δ6, Δ8, Δ10, Δ12, Δ14, Δ16 and Δ18 amino acid deletions. (C) The modified full length RSV F gene (#683) with a modified cleavage site II (KKQKQQ) and Phe137 - Ser146 fusion domain deletion including RSV F cleavable signal peptide (sp), 27 amino acid peptide [23] , hydrophobic transmembrane (tm), and F2 and F1 polypeptides covalently linked by two disulfides.

    Techniques Used: Mutagenesis, Modification

    61) Product Images from "Identification of an Imprinted Gene Cluster in the X-Inactivation Center"

    Article Title: Identification of an Imprinted Gene Cluster in the X-Inactivation Center

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071222

    Identification of small RNAs predominantly expressed in female blastocysts. (A) Scheme of the X GFP sexing system and construction of small RNA libraries. Transgenic male mice (X GFP Y) were mated with wild-type female mice (XX). Only the female embryos (XX GFP ) fluoresce green because of the paternally inherited X GFP . Small RNAs obtained from male and female blastocysts were sequenced and expression levels were compared between sexes. (B) Annotation of the read sequences from male and female blastocyst libraries. The distribution of the RNA classes is given as a percentage of the total annotated ncRNAs. (C) Expression levels of miR-374-5p, miR-421-3p and miR-295 miRNA in male and female blastocysts. The expression of three miRNAs was measured by qRT–PCR and normalized to that of snoRNA202 examined as a control. The data indicate that the miR-374-5p and miR-421-3p were predominantly expressed in female blastocysts (* P
    Figure Legend Snippet: Identification of small RNAs predominantly expressed in female blastocysts. (A) Scheme of the X GFP sexing system and construction of small RNA libraries. Transgenic male mice (X GFP Y) were mated with wild-type female mice (XX). Only the female embryos (XX GFP ) fluoresce green because of the paternally inherited X GFP . Small RNAs obtained from male and female blastocysts were sequenced and expression levels were compared between sexes. (B) Annotation of the read sequences from male and female blastocyst libraries. The distribution of the RNA classes is given as a percentage of the total annotated ncRNAs. (C) Expression levels of miR-374-5p, miR-421-3p and miR-295 miRNA in male and female blastocysts. The expression of three miRNAs was measured by qRT–PCR and normalized to that of snoRNA202 examined as a control. The data indicate that the miR-374-5p and miR-421-3p were predominantly expressed in female blastocysts (* P

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Quantitative RT-PCR

    Verification of imprinting of two miRNAs predominantly expressed in female embryos. (A) Schematic representation of two mature miRNAs, miR-374-5p and miR-421-3p and their preceding transcript pri-miRNA. Both miRNAs were clustered and located in the intron of the Ftx (B230206F22Rik) gene on the X chromosome. Both are thought to be transcribed as pri-miRNAs and then processed to form mature miRNAs. The location of the primers used to assess the pri-miRNA and Ftx expression levels are indicated: qRT–PCR primers 207 and 208 were used for Ftx ; primers used for allelic discrimination were 057 and 058 for pri-miRNA, 116 and 117 for Ftx . The exons for Ftx are numbered according to a previous report [22] . DNA polymorphisms used for allelic discrimination are indicated with red characters. PCR primers correspond to the list in Table S3 (B) Production of mature miRNAs from pre-miRNAs in female blastocysts. Although two mature forms of miRNA, miR-374-5p/miR-374-3p and miR-421-5p/miR-421-3p are registered in miRBase, only miR-374-5p and miR-421-3p were detected at the blastocyst stage. Orange coloring shows mismatch sequences compared with the reference sequences, suggesting miRNA editing or misreading by the 454 sequencer. (C) Expression of pri-miRNA in wild-type male and female blastocysts obtained from the uterus. Female-predominant expression was observed as well as previously reported X-linked imprinted genes such as Xist and Rhox5 . (D) Verification of imprinting of the pri-miRNA for miR-374-5p and miR-421-3p in preimplantation mouse embryos. Pri-miRNA was expressed from the paternal B6 allele in JF1× C57BL/6 crosses and from the paternal JF1 allele in the reciprocal B6× (JF1× B6) cross. The arrows indicate the polymorphism sites that were used in this assay.
    Figure Legend Snippet: Verification of imprinting of two miRNAs predominantly expressed in female embryos. (A) Schematic representation of two mature miRNAs, miR-374-5p and miR-421-3p and their preceding transcript pri-miRNA. Both miRNAs were clustered and located in the intron of the Ftx (B230206F22Rik) gene on the X chromosome. Both are thought to be transcribed as pri-miRNAs and then processed to form mature miRNAs. The location of the primers used to assess the pri-miRNA and Ftx expression levels are indicated: qRT–PCR primers 207 and 208 were used for Ftx ; primers used for allelic discrimination were 057 and 058 for pri-miRNA, 116 and 117 for Ftx . The exons for Ftx are numbered according to a previous report [22] . DNA polymorphisms used for allelic discrimination are indicated with red characters. PCR primers correspond to the list in Table S3 (B) Production of mature miRNAs from pre-miRNAs in female blastocysts. Although two mature forms of miRNA, miR-374-5p/miR-374-3p and miR-421-5p/miR-421-3p are registered in miRBase, only miR-374-5p and miR-421-3p were detected at the blastocyst stage. Orange coloring shows mismatch sequences compared with the reference sequences, suggesting miRNA editing or misreading by the 454 sequencer. (C) Expression of pri-miRNA in wild-type male and female blastocysts obtained from the uterus. Female-predominant expression was observed as well as previously reported X-linked imprinted genes such as Xist and Rhox5 . (D) Verification of imprinting of the pri-miRNA for miR-374-5p and miR-421-3p in preimplantation mouse embryos. Pri-miRNA was expressed from the paternal B6 allele in JF1× C57BL/6 crosses and from the paternal JF1 allele in the reciprocal B6× (JF1× B6) cross. The arrows indicate the polymorphism sites that were used in this assay.

    Techniques Used: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction

    Allelic expression analysis of female-expressed genes at the blastocyst stage. (A) Verification of imprinting in the Ftx gene. Upper: schematic representation of the position of the DNA polymorphism in the Ftx gene. The single nucleotide polymorphism detected in C57BL/6: B6 (aCtgt) and JF1/Ms: JF1 (aTtgt) is shown. The Hpy CH4III site in the B6 allele was changed in the JF1 allele. These alleles could be distinguished by digestion with Hpy CH4III. Lower: the imprinted expression of Ftx from the Xp chromosome was determined by qRT–PCR and restriction fragment length polymorphism (RFLP) analysis with inter-subspecific hybrid mouse F1 progenies. (B) Verification of Jpx imprinting. Upper: schematic representation of the position of the DNA polymorphism in the Jpx gene. The Sty I site in the B6 allele (ccaaAg) was changed in the JF1 allele (to ccaaGg). Lower: imprinted expression of Jpx from the Xp chromosome was determined by qRT–PCR and RFLP analysis with inter-subspecific hybrid mouse F1 progenies. (C) Verification of the Zcchc13 imprinting. The single nucleotide insertion polymorphism detected in C57BL/6; B6 (triple A) and JF1/Ms; and JF1 (double A) is shown. The imprinted expression of Zcchc13 from the Xp chromosome was determined by sequencing analysis with inter-subspecific hybrid mice F1 progenies. (D) Whole-mount in situ hybridization of the Ftx gene in female and male blastocysts. ‘S’ indicates the sense strand probe and ‘A’ indicates the antisense strand probe. Abbreviation: ICM, Inner cell mass; TE, trophectoderm.
    Figure Legend Snippet: Allelic expression analysis of female-expressed genes at the blastocyst stage. (A) Verification of imprinting in the Ftx gene. Upper: schematic representation of the position of the DNA polymorphism in the Ftx gene. The single nucleotide polymorphism detected in C57BL/6: B6 (aCtgt) and JF1/Ms: JF1 (aTtgt) is shown. The Hpy CH4III site in the B6 allele was changed in the JF1 allele. These alleles could be distinguished by digestion with Hpy CH4III. Lower: the imprinted expression of Ftx from the Xp chromosome was determined by qRT–PCR and restriction fragment length polymorphism (RFLP) analysis with inter-subspecific hybrid mouse F1 progenies. (B) Verification of Jpx imprinting. Upper: schematic representation of the position of the DNA polymorphism in the Jpx gene. The Sty I site in the B6 allele (ccaaAg) was changed in the JF1 allele (to ccaaGg). Lower: imprinted expression of Jpx from the Xp chromosome was determined by qRT–PCR and RFLP analysis with inter-subspecific hybrid mouse F1 progenies. (C) Verification of the Zcchc13 imprinting. The single nucleotide insertion polymorphism detected in C57BL/6; B6 (triple A) and JF1/Ms; and JF1 (double A) is shown. The imprinted expression of Zcchc13 from the Xp chromosome was determined by sequencing analysis with inter-subspecific hybrid mice F1 progenies. (D) Whole-mount in situ hybridization of the Ftx gene in female and male blastocysts. ‘S’ indicates the sense strand probe and ‘A’ indicates the antisense strand probe. Abbreviation: ICM, Inner cell mass; TE, trophectoderm.

    Techniques Used: Expressing, Mass Spectrometry, Quantitative RT-PCR, Sequencing, Mouse Assay, In Situ Hybridization

    Searching for a cluster of imprinted genes on the X chromosome. (A) qRT–PCR of X-linked genes in male and female blastocysts. It should be noted that very weak expression levels of Xist, Jpx, Ftx, Zcchc13 were detected in male blastocysts when additional PCR cycles were carried out. (B) The expression levels of Chic , Tsx , Xite, Tsix, Xist , Jpx , Ftx , Zcchc13 , Rlim1/Rnf12 , Slc16a2 , C77370 were quantified by qRT–PCR in female and male mouse blastocysts. The ratios of the number of cDNA copies for each gene to that of cDNA copies for a housekeeping gene, β-actin , are indicated as expression levels. Expressions of Xite and Scl16a2 were not detected at the blastocyst stage (data not shown). Results are expressed as the mean ± SD ( n = 3). The statistical significance of differences between sample means was determined by Student’s t -test. * P
    Figure Legend Snippet: Searching for a cluster of imprinted genes on the X chromosome. (A) qRT–PCR of X-linked genes in male and female blastocysts. It should be noted that very weak expression levels of Xist, Jpx, Ftx, Zcchc13 were detected in male blastocysts when additional PCR cycles were carried out. (B) The expression levels of Chic , Tsx , Xite, Tsix, Xist , Jpx , Ftx , Zcchc13 , Rlim1/Rnf12 , Slc16a2 , C77370 were quantified by qRT–PCR in female and male mouse blastocysts. The ratios of the number of cDNA copies for each gene to that of cDNA copies for a housekeeping gene, β-actin , are indicated as expression levels. Expressions of Xite and Scl16a2 were not detected at the blastocyst stage (data not shown). Results are expressed as the mean ± SD ( n = 3). The statistical significance of differences between sample means was determined by Student’s t -test. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Polymerase Chain Reaction

    62) Product Images from "Identification of an Imprinted Gene Cluster in the X-Inactivation Center"

    Article Title: Identification of an Imprinted Gene Cluster in the X-Inactivation Center

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071222

    Searching for a cluster of imprinted genes on the X chromosome. (A) qRT–PCR of X-linked genes in male and female blastocysts. It should be noted that very weak expression levels of Xist, Jpx, Ftx, Zcchc13 were detected in male blastocysts when additional PCR cycles were carried out. (B) The expression levels of Chic , Tsx , Xite, Tsix, Xist , Jpx , Ftx , Zcchc13 , Rlim1/Rnf12 , Slc16a2 , C77370 were quantified by qRT–PCR in female and male mouse blastocysts. The ratios of the number of cDNA copies for each gene to that of cDNA copies for a housekeeping gene, β-actin , are indicated as expression levels. Expressions of Xite and Scl16a2 were not detected at the blastocyst stage (data not shown). Results are expressed as the mean ± SD ( n = 3). The statistical significance of differences between sample means was determined by Student’s t -test. * P
    Figure Legend Snippet: Searching for a cluster of imprinted genes on the X chromosome. (A) qRT–PCR of X-linked genes in male and female blastocysts. It should be noted that very weak expression levels of Xist, Jpx, Ftx, Zcchc13 were detected in male blastocysts when additional PCR cycles were carried out. (B) The expression levels of Chic , Tsx , Xite, Tsix, Xist , Jpx , Ftx , Zcchc13 , Rlim1/Rnf12 , Slc16a2 , C77370 were quantified by qRT–PCR in female and male mouse blastocysts. The ratios of the number of cDNA copies for each gene to that of cDNA copies for a housekeeping gene, β-actin , are indicated as expression levels. Expressions of Xite and Scl16a2 were not detected at the blastocyst stage (data not shown). Results are expressed as the mean ± SD ( n = 3). The statistical significance of differences between sample means was determined by Student’s t -test. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Polymerase Chain Reaction

    63) Product Images from "TgpA, a Protein with a Eukaryotic-Like Transglutaminase Domain, Plays a Critical Role in the Viability of Pseudomonas aeruginosa"

    Article Title: TgpA, a Protein with a Eukaryotic-Like Transglutaminase Domain, Plays a Critical Role in the Viability of Pseudomonas aeruginosa

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0050323

    Genetic organization and transcription analysis of the genomic region including PA2873 locus ( tgpA ). (A) PA2875-2874-2873-2872 gene cluster is represented according to visualization by GBrowse in Pseudomonas Genome Database. Locations of fragments amplified by the oligo pairs (Roman numbers) used in RT-PCR-based transcription analysis (B) are shown along the region map. The position of plasmid pDM4 cointegration in PAO1 PA2875::pDM4 strain is indicated. (B) Total RNA was extracted from PAO1 and PAO1 PA2875::pDM4 cells in both exponential (E) and stationary (S) phases, and analyzed by RT-PCR. RT untreated samples (RT−) as controls of genomic DNA contamination were included in the analysis.
    Figure Legend Snippet: Genetic organization and transcription analysis of the genomic region including PA2873 locus ( tgpA ). (A) PA2875-2874-2873-2872 gene cluster is represented according to visualization by GBrowse in Pseudomonas Genome Database. Locations of fragments amplified by the oligo pairs (Roman numbers) used in RT-PCR-based transcription analysis (B) are shown along the region map. The position of plasmid pDM4 cointegration in PAO1 PA2875::pDM4 strain is indicated. (B) Total RNA was extracted from PAO1 and PAO1 PA2875::pDM4 cells in both exponential (E) and stationary (S) phases, and analyzed by RT-PCR. RT untreated samples (RT−) as controls of genomic DNA contamination were included in the analysis.

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

    64) Product Images from "5-Lipoxygenase DNA Methylation and mRNA Content in the Brain and Heart of Young and Old Mice"

    Article Title: 5-Lipoxygenase DNA Methylation and mRNA Content in the Brain and Heart of Young and Old Mice

    Journal: Neural Plasticity

    doi: 10.1155/2009/209596

    The sequence of the mouse 5-LOX promoter-5′ UTR and the first exon-intron region subjected to the methylation-sensitive endonuclease assay with the four endonucleases—AciI, BstUI, HinP1I, and HpaII. The yellow highlight indicates the first exon including the start codon (ATG, in red). The four endonuclease-targeted methylation-sensitive endonuclease recognition sites are highlighted by four different colors. The selected DNA sequence included 2 AciI-sensitive sites (upstream of the start codon), 6 BstUI-sensitive sites (2 upstream and 4 downstream of the start codon), 8 HinP1I-sensitive sites (2 upstream and 6 downstream of the start codon), and 3 HpaII-sensitive sites (2 upstream and 1 downstream of the start codon). The primer regions used for quantitative PCR amplification are underlined; primers were used as follows. Promoter - 5′ UTR: forward = 5′-aga gaa gga tgc gtt gga aggt-3′ and reverse = 5′-gac tcc ggg caa gtg agt gct-3′; exon-intron: forward = 5′-agt cat gcc ctc cta cac ggt ca-3′ and reverse = 5′-agt cat gcc ctc cta cac ggt ca-3′. For the input control, we used the following primers: forward = 5′-tga tgt ggc tgg cct ctt atg tga-3′, reverse = 5′-act ggg act gag tgc agg aaa tgt-3′.
    Figure Legend Snippet: The sequence of the mouse 5-LOX promoter-5′ UTR and the first exon-intron region subjected to the methylation-sensitive endonuclease assay with the four endonucleases—AciI, BstUI, HinP1I, and HpaII. The yellow highlight indicates the first exon including the start codon (ATG, in red). The four endonuclease-targeted methylation-sensitive endonuclease recognition sites are highlighted by four different colors. The selected DNA sequence included 2 AciI-sensitive sites (upstream of the start codon), 6 BstUI-sensitive sites (2 upstream and 4 downstream of the start codon), 8 HinP1I-sensitive sites (2 upstream and 6 downstream of the start codon), and 3 HpaII-sensitive sites (2 upstream and 1 downstream of the start codon). The primer regions used for quantitative PCR amplification are underlined; primers were used as follows. Promoter - 5′ UTR: forward = 5′-aga gaa gga tgc gtt gga aggt-3′ and reverse = 5′-gac tcc ggg caa gtg agt gct-3′; exon-intron: forward = 5′-agt cat gcc ctc cta cac ggt ca-3′ and reverse = 5′-agt cat gcc ctc cta cac ggt ca-3′. For the input control, we used the following primers: forward = 5′-tga tgt ggc tgg cct ctt atg tga-3′, reverse = 5′-act ggg act gag tgc agg aaa tgt-3′.

    Techniques Used: Sequencing, Methylation, Real-time Polymerase Chain Reaction, Amplification, Cellular Antioxidant Activity Assay, Activated Clotting Time Assay

    An example of the SYBR Green RD-qPCR assay of 5-LOX DNA methylation (shown are graphs obtained from 10 cerebellar samples). The samples were digested with the methylation-sensitive endonucleases (AciI, BstUI, HinP1I, and HpaII) as described in Material and Methods. The PCR reaction for the promoter-5′UTR (10 samples each: AciI = blue, BstUI = red, HinP1I = green, and HpaII = gray) and corresponding input control regions (shown in yellow) were carried out in separate tubes. Panel A shows the dissociation curve data, which indicate the presence of only one PCR product (peak) for each specific set of primers (fluorescence (first derivative of the raw fluorescence reading multiplied by −1) on the Y-axis versus the PCR product melting temperature (°C) on the X-axis). Panel B shows examples of the amplification plots used for calculating the quantitative data (the amplification plots fluorescence (baseline-corrected raw fluorescence) on the Y-axis versus cycle number on the X-axis). In this assay, the threshold cycle is inversely proportional to the log of the initial copy number. In other words, the more template that is present initially, the fewer the number of cycles required for the fluorescence signal to be detectable above background.
    Figure Legend Snippet: An example of the SYBR Green RD-qPCR assay of 5-LOX DNA methylation (shown are graphs obtained from 10 cerebellar samples). The samples were digested with the methylation-sensitive endonucleases (AciI, BstUI, HinP1I, and HpaII) as described in Material and Methods. The PCR reaction for the promoter-5′UTR (10 samples each: AciI = blue, BstUI = red, HinP1I = green, and HpaII = gray) and corresponding input control regions (shown in yellow) were carried out in separate tubes. Panel A shows the dissociation curve data, which indicate the presence of only one PCR product (peak) for each specific set of primers (fluorescence (first derivative of the raw fluorescence reading multiplied by −1) on the Y-axis versus the PCR product melting temperature (°C) on the X-axis). Panel B shows examples of the amplification plots used for calculating the quantitative data (the amplification plots fluorescence (baseline-corrected raw fluorescence) on the Y-axis versus cycle number on the X-axis). In this assay, the threshold cycle is inversely proportional to the log of the initial copy number. In other words, the more template that is present initially, the fewer the number of cycles required for the fluorescence signal to be detectable above background.

    Techniques Used: SYBR Green Assay, Real-time Polymerase Chain Reaction, DNA Methylation Assay, Methylation, Polymerase Chain Reaction, Fluorescence, Amplification

    65) Product Images from "Systematic Identification of mRNAs Recruited to Argonaute 2 by Specific microRNAs and Corresponding Changes in Transcript Abundance"

    Article Title: Systematic Identification of mRNAs Recruited to Argonaute 2 by Specific microRNAs and Corresponding Changes in Transcript Abundance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002126

    Comparison of expression changes of mRNAs containing seed matches in 3′-UTRs and coding sequences of miR-1 and miR-124 Ago2 IP targets. (A) Cumulative distribution of the change in mRNA levels following transfection with FLAG-Ago2 and miR-1 compared to FLAG-Ago2 alone. This analysis included Ago2 IP targets with 3′-UTR 7mer seed matches, but no coding sequence 6mer seed matches (21, red), Ago2 IP targets with coding sequence 7mer seed matches, but no 3′-UTR 6mer seed matches (10, green), and mRNAs that did not contain 3′-UTR or coding sequence 6mer seed matches (2893, black). Changes in mRNA levels of Ago2 IP targets with 3′-UTR 7mer seed matches were greater than those for Ago2 IP targets with coding sequence 7mer seed matches (P = 0.0004), which were in turn greater than those for mRNAs without any 6mer seed matches in the 3′-UTR or coding sequence (P = 0.006). (B) Same as in (A) except for mRNAs associated with FLAG-Ago2 upon transfection with miR-124. There were 81 Ago2 IP targets with 3′-UTR 7mer seed matches but no 6mer coding sequence seed matches (red), 43 Ago2 IP targets with coding sequence 7mer seed matches but no 6mer 3′-UTR seed matches (green), and 1877 mRNAs with no 6mer seed matches in their 3′-UTR or coding sequence. Changes in mRNA levels of Ago2 IP targets with 3′-UTR 7mer seed matches were greater than the changes for Ago2 IP targets with coding sequence 7mer seed matches (P = 0.0005), which in turn were greater than the changes for mRNAs without any 6mer seed matches in the 3′-UTR or coding sequence (P
    Figure Legend Snippet: Comparison of expression changes of mRNAs containing seed matches in 3′-UTRs and coding sequences of miR-1 and miR-124 Ago2 IP targets. (A) Cumulative distribution of the change in mRNA levels following transfection with FLAG-Ago2 and miR-1 compared to FLAG-Ago2 alone. This analysis included Ago2 IP targets with 3′-UTR 7mer seed matches, but no coding sequence 6mer seed matches (21, red), Ago2 IP targets with coding sequence 7mer seed matches, but no 3′-UTR 6mer seed matches (10, green), and mRNAs that did not contain 3′-UTR or coding sequence 6mer seed matches (2893, black). Changes in mRNA levels of Ago2 IP targets with 3′-UTR 7mer seed matches were greater than those for Ago2 IP targets with coding sequence 7mer seed matches (P = 0.0004), which were in turn greater than those for mRNAs without any 6mer seed matches in the 3′-UTR or coding sequence (P = 0.006). (B) Same as in (A) except for mRNAs associated with FLAG-Ago2 upon transfection with miR-124. There were 81 Ago2 IP targets with 3′-UTR 7mer seed matches but no 6mer coding sequence seed matches (red), 43 Ago2 IP targets with coding sequence 7mer seed matches but no 6mer 3′-UTR seed matches (green), and 1877 mRNAs with no 6mer seed matches in their 3′-UTR or coding sequence. Changes in mRNA levels of Ago2 IP targets with 3′-UTR 7mer seed matches were greater than the changes for Ago2 IP targets with coding sequence 7mer seed matches (P = 0.0005), which in turn were greater than the changes for mRNAs without any 6mer seed matches in the 3′-UTR or coding sequence (P

    Techniques Used: Expressing, Transfection, Sequencing

    66) Product Images from "Nuclear Import and DNA Binding of the ZHD5 Transcription Factor Is Modulated by a Competitive Peptide Inhibitor in Arabidopsis *"

    Article Title: Nuclear Import and DNA Binding of the ZHD5 Transcription Factor Is Modulated by a Competitive Peptide Inhibitor in Arabidopsis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.167692

    Phenotypic recovery of transgenic plants overexpressing ZHD5 by MIF1 coexpression. A , phenotypes of transgenic plants. The MIF1 or ZHD5 gene was overexpressed under the control of the CaMV 35 S promoter. Five-week-old transgenic plants grown in soil were photographed. The MIF1 x ZHD5 plants were produced by genetic cross of the 35S: MIF1 and 35S: ZHD5 transgenic plants. The inlet shows an enlarged view of the 35S: MIF1 transgenic plant. B , expression of the MIF1 and ZHD5 genes in the transgenic plants. Transcript levels were determined by qRT-PCR using total RNA samples extracted from 2-week-old, whole plants grown on MS-agar plates. Biological triplicates were averaged. Error bars , S.E. Statistical significance was determined using Student's t test (*, p
    Figure Legend Snippet: Phenotypic recovery of transgenic plants overexpressing ZHD5 by MIF1 coexpression. A , phenotypes of transgenic plants. The MIF1 or ZHD5 gene was overexpressed under the control of the CaMV 35 S promoter. Five-week-old transgenic plants grown in soil were photographed. The MIF1 x ZHD5 plants were produced by genetic cross of the 35S: MIF1 and 35S: ZHD5 transgenic plants. The inlet shows an enlarged view of the 35S: MIF1 transgenic plant. B , expression of the MIF1 and ZHD5 genes in the transgenic plants. Transcript levels were determined by qRT-PCR using total RNA samples extracted from 2-week-old, whole plants grown on MS-agar plates. Biological triplicates were averaged. Error bars , S.E. Statistical significance was determined using Student's t test (*, p

    Techniques Used: Transgenic Assay, Produced, Expressing, Quantitative RT-PCR, Mass Spectrometry

    67) Product Images from "Formate hydrogenlyase in the hyperthermophilic archaeon, Thermococcus litoralis"

    Article Title: Formate hydrogenlyase in the hyperthermophilic archaeon, Thermococcus litoralis

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-8-88

    Gene expression studies with reverse transcription linked absolute quantification Real-Time PCR and immunoblotting . A . Relative transcription level of fdh-mhy operon in cells grown on different carbon sources (M and P denotes for the maltose and peptide (casein hydrolysate) content of the medium, respectively, see Materials and Methods). The transcription level of the fdh-mhy operon in cells grown on DP medium was taken as 100 %. For reverse transcription RNA was purified from three parallel cultures, cDNA was made using the fhl8frC1R primer designed to the 3' end of mhyH . In the Real-Time PCR a primer pair (fhl10N-fhl05R) designed to the mhyF gene was used. B . Effect of sulfur on the transcription level of fdh-mhy operon (M, P and S denotes for the maltose, peptides (casein hydrolysate) and sulfur content of the medium, respectively, see Materials and Methods). For reverse transcription RNA was purified from three parallel cultures, cDNA was made with fhl805R primer designed to the 3' end of mhyC . In the Real-Time PCR a primer pair (fhl805R-fhl806N) designed to the mhyC was used. C. D . Western blotting of the same samples as in A (C) and B (D). In the immunoblotting experiments 20 μg total cellular protein, derived from the same cultures as above, was separated on 12% SDS polyacrylamide gel after boiling in SDS-loading buffer for 10 min. Proteins were blotted on nitrocellulose membrane and the FdhB protein was detected using anti-FdhB antibody.
    Figure Legend Snippet: Gene expression studies with reverse transcription linked absolute quantification Real-Time PCR and immunoblotting . A . Relative transcription level of fdh-mhy operon in cells grown on different carbon sources (M and P denotes for the maltose and peptide (casein hydrolysate) content of the medium, respectively, see Materials and Methods). The transcription level of the fdh-mhy operon in cells grown on DP medium was taken as 100 %. For reverse transcription RNA was purified from three parallel cultures, cDNA was made using the fhl8frC1R primer designed to the 3' end of mhyH . In the Real-Time PCR a primer pair (fhl10N-fhl05R) designed to the mhyF gene was used. B . Effect of sulfur on the transcription level of fdh-mhy operon (M, P and S denotes for the maltose, peptides (casein hydrolysate) and sulfur content of the medium, respectively, see Materials and Methods). For reverse transcription RNA was purified from three parallel cultures, cDNA was made with fhl805R primer designed to the 3' end of mhyC . In the Real-Time PCR a primer pair (fhl805R-fhl806N) designed to the mhyC was used. C. D . Western blotting of the same samples as in A (C) and B (D). In the immunoblotting experiments 20 μg total cellular protein, derived from the same cultures as above, was separated on 12% SDS polyacrylamide gel after boiling in SDS-loading buffer for 10 min. Proteins were blotted on nitrocellulose membrane and the FdhB protein was detected using anti-FdhB antibody.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Purification, Western Blot, Derivative Assay

    68) Product Images from "Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA"

    Article Title: Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA

    Journal: Molecular Microbiology

    doi: 10.1111/mmi.12358

    HigB expression in M. smegmatis .A. Genomic organization of the M. tuberculosis HigBA locus.B and C. M. smegmatis strains carrying vector control (‘Vector’) or the HigB expression plasmid (‘HigB’) grown in the absence (−) or presence (+) of anhydrotetracycline (ATc). (B) Quantitative RT-PCR of higB expression relative to 16S rRNA. RNA was isolated from exponential-phase cultures. (C) Growth of M. smegmatis strains. Liquid cultures of transformants were grown from a starting OD 580 of 0.05 and monitored over time. No or 300 ng ml −1 ATc were added at the start of the growth curve. All results are the mean values and standard deviation of three independent biological replicates. Where indicated, a significant difference (as determined by Student's t -test) between uninduced (− ATc) and induced (+ ATc) conditions is marked by an asterisk (*) for P
    Figure Legend Snippet: HigB expression in M. smegmatis .A. Genomic organization of the M. tuberculosis HigBA locus.B and C. M. smegmatis strains carrying vector control (‘Vector’) or the HigB expression plasmid (‘HigB’) grown in the absence (−) or presence (+) of anhydrotetracycline (ATc). (B) Quantitative RT-PCR of higB expression relative to 16S rRNA. RNA was isolated from exponential-phase cultures. (C) Growth of M. smegmatis strains. Liquid cultures of transformants were grown from a starting OD 580 of 0.05 and monitored over time. No or 300 ng ml −1 ATc were added at the start of the growth curve. All results are the mean values and standard deviation of three independent biological replicates. Where indicated, a significant difference (as determined by Student's t -test) between uninduced (− ATc) and induced (+ ATc) conditions is marked by an asterisk (*) for P

    Techniques Used: Expressing, Plasmid Preparation, Quantitative RT-PCR, Isolation, Standard Deviation

    HigB expression affects tmRNA in M. tuberculosis ΔTAC. RNA was extracted from mid-exponential cultures treated with ATc for 2 h, 6 h or 24 h and cDNA was prepared for qRT-PCR analysis.A–C. Northern blots probing for tmRNA (A and B) and 5S (C). Transcript sizes are relative to the position of RNA marker and tmRNA cleavage products are indicated with an arrow.D. Quantitative RT-PCR of transcripts of interest. All results are the mean values and standard deviation of three independent biological replicates. A significant difference (as determined by Student's t -test) between vector control and HigB overexpression strain is marked by an asterisk (*) for P
    Figure Legend Snippet: HigB expression affects tmRNA in M. tuberculosis ΔTAC. RNA was extracted from mid-exponential cultures treated with ATc for 2 h, 6 h or 24 h and cDNA was prepared for qRT-PCR analysis.A–C. Northern blots probing for tmRNA (A and B) and 5S (C). Transcript sizes are relative to the position of RNA marker and tmRNA cleavage products are indicated with an arrow.D. Quantitative RT-PCR of transcripts of interest. All results are the mean values and standard deviation of three independent biological replicates. A significant difference (as determined by Student's t -test) between vector control and HigB overexpression strain is marked by an asterisk (*) for P

    Techniques Used: Expressing, Quantitative RT-PCR, Northern Blot, Marker, Standard Deviation, Plasmid Preparation, Over Expression

    HigB expression in M. tuberculosis. M. tuberculosis strains carrying vector control (‘Vector’) or the HigB expression plasmid (‘HigB’) grown in the absence (−) or presence (+) of ATc.A. Quantitative RT-PCR of higB expression relative to 16S rRNA. RNA was isolated from M. tuberculosis wild-type exponential-phase cultures.B–D. Growth of M. tuberculosis wild-type (B) and ΔTAC (C and D) strains. Liquid cultures of transformants were grown from a starting OD 580 of 0.05 and monitored over time. No or 300 ng ml −1 ATc were added at the start of the growth curve at day 0 (B and C) or during mid-exponential phase at day 3 (D).E. Survival of ΔTAC transformants following addition of ATc during mid-exponential phase (time 0) and removal of inducer by washing at day 7.All results are the mean values and standard deviation of three independent biological replicates. A significant difference (as determined by Student's t -test) between uninduced (− ATc) and induced (+ ATc) conditions is marked by an asterisk (*) for P
    Figure Legend Snippet: HigB expression in M. tuberculosis. M. tuberculosis strains carrying vector control (‘Vector’) or the HigB expression plasmid (‘HigB’) grown in the absence (−) or presence (+) of ATc.A. Quantitative RT-PCR of higB expression relative to 16S rRNA. RNA was isolated from M. tuberculosis wild-type exponential-phase cultures.B–D. Growth of M. tuberculosis wild-type (B) and ΔTAC (C and D) strains. Liquid cultures of transformants were grown from a starting OD 580 of 0.05 and monitored over time. No or 300 ng ml −1 ATc were added at the start of the growth curve at day 0 (B and C) or during mid-exponential phase at day 3 (D).E. Survival of ΔTAC transformants following addition of ATc during mid-exponential phase (time 0) and removal of inducer by washing at day 7.All results are the mean values and standard deviation of three independent biological replicates. A significant difference (as determined by Student's t -test) between uninduced (− ATc) and induced (+ ATc) conditions is marked by an asterisk (*) for P

    Techniques Used: Expressing, Plasmid Preparation, Quantitative RT-PCR, Isolation, Standard Deviation

    HigB expression affects tmRNA in M. smegmatis and E. coli .A. DNA alignment of ssrA from M. tuberculosis and E. coli . Conserved residues are highlighted by an asterisk (*).B. Northern blots probing for tmRNA (B) and 5 S (C). RNA was extracted from M. smegmatis (left panel) and E. coli (right panel) HigB expression strains grown with or without inducer. Strains were grown to mid-exponential phase and inducer was added; ATc for M. smegmatis and l -arabinose for E. coli , for 7.5 h and 2 h respectively.
    Figure Legend Snippet: HigB expression affects tmRNA in M. smegmatis and E. coli .A. DNA alignment of ssrA from M. tuberculosis and E. coli . Conserved residues are highlighted by an asterisk (*).B. Northern blots probing for tmRNA (B) and 5 S (C). RNA was extracted from M. smegmatis (left panel) and E. coli (right panel) HigB expression strains grown with or without inducer. Strains were grown to mid-exponential phase and inducer was added; ATc for M. smegmatis and l -arabinose for E. coli , for 7.5 h and 2 h respectively.

    Techniques Used: Expressing, Northern Blot

    69) Product Images from "Expression and Function of Ephrin-B1 and Its Cognate Receptor EphB2 in Human Abdominal Aortic Aneurysm"

    Article Title: Expression and Function of Ephrin-B1 and Its Cognate Receptor EphB2 in Human Abdominal Aortic Aneurysm

    Journal: International Journal of Vascular Medicine

    doi: 10.1155/2012/127149

    RT-PCR analysis for ephrin-B1 and EphB2 in human PBMC. Three kinds of templates were used to validate the results: lane 1, cDNA; lane 2, DNA-cleared RNA; lane 3; genomic DNA.
    Figure Legend Snippet: RT-PCR analysis for ephrin-B1 and EphB2 in human PBMC. Three kinds of templates were used to validate the results: lane 1, cDNA; lane 2, DNA-cleared RNA; lane 3; genomic DNA.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    70) Product Images from "HCMV Protein LUNA Is Required for Viral Reactivation from Latently Infected Primary CD14+ Cells"

    Article Title: HCMV Protein LUNA Is Required for Viral Reactivation from Latently Infected Primary CD14+ Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052827

    LUNA protein expression is not required for lytic gene expression, but does augment the expression of the UL138 transcript. HF cells infected at an MOI = 1 with either FIX-WT, FIX-ΔLUNA and FIX-Rev were harvested at the indicated time points for either RNA or protein analysis. A) Expression of viral RNAs. Total RNA was collected over a 20 d time course together with mock RNA. From all the collected RNA samples cDNA was synthesized and amplified using UL82, UL123, UL81-82ast, UL138 and β-actin specific primers. Negative images were used to visualize weaker bands. B) qRT-PCR analysis was used to assay gene expression of UL138 at the indicated times post infection. Viral mRNA was normalized to actin. Asterisks indicate significant changes (p value
    Figure Legend Snippet: LUNA protein expression is not required for lytic gene expression, but does augment the expression of the UL138 transcript. HF cells infected at an MOI = 1 with either FIX-WT, FIX-ΔLUNA and FIX-Rev were harvested at the indicated time points for either RNA or protein analysis. A) Expression of viral RNAs. Total RNA was collected over a 20 d time course together with mock RNA. From all the collected RNA samples cDNA was synthesized and amplified using UL82, UL123, UL81-82ast, UL138 and β-actin specific primers. Negative images were used to visualize weaker bands. B) qRT-PCR analysis was used to assay gene expression of UL138 at the indicated times post infection. Viral mRNA was normalized to actin. Asterisks indicate significant changes (p value

    Techniques Used: Expressing, Infection, Synthesized, Amplification, Quantitative RT-PCR, Significance Assay

    FIX-ΔLUNA infected CD14+ cells fail to express lytic transcripts following IL6 induced differentiation. A) Diagram of the timeline of infection. Infected cells were collected at 1–20 dpi, IL6 was added at 10 dpi to induce cellular differentiation, after which two additional time points were collected. B) Expression of viral RNAs in FIX-WT, FIX-ΔLUNA and FIX-Rev infected CD14 + cells (MOI = 1). Total RNA was collected over a 20 d time course together with mock RNA. At 10 dpi CD14 + cells were differentiated with IL-6. Five and ten days post differentiation, RNA samples were collected (these samples were termed as 15 d-IL6 and 20 d-IL6). From all the collected RNA samples cDNA was synthesized and amplified using UL82, UL123, UL81-82ast, UL138 and β-actin specific primers. Negative images were used to visualize weaker bands. C) CD14 + cells were infected at an MOI = 1 with FIX-WT, FIX-ΔLUNA or FIX-Rev. D–E) Protein was isolated from infected CD14 + cells at the indicated time points and subjected to western blot analysis. Blots were probed with either a rabbit monoclonal a-LUNA or a mouse monoclonal a-IE1 antibody along with a-Tubulin as a loading control. qRT-PCR analysis was used to assay gene expression of UL138, vIL10 and US28 at the indicated times post infection. Viral mRNA was normalized to actin. All samples were tested in triplicate. Abbreviations used: d, days post infection., Mo; mock. IL6 was added at 10 dpi.
    Figure Legend Snippet: FIX-ΔLUNA infected CD14+ cells fail to express lytic transcripts following IL6 induced differentiation. A) Diagram of the timeline of infection. Infected cells were collected at 1–20 dpi, IL6 was added at 10 dpi to induce cellular differentiation, after which two additional time points were collected. B) Expression of viral RNAs in FIX-WT, FIX-ΔLUNA and FIX-Rev infected CD14 + cells (MOI = 1). Total RNA was collected over a 20 d time course together with mock RNA. At 10 dpi CD14 + cells were differentiated with IL-6. Five and ten days post differentiation, RNA samples were collected (these samples were termed as 15 d-IL6 and 20 d-IL6). From all the collected RNA samples cDNA was synthesized and amplified using UL82, UL123, UL81-82ast, UL138 and β-actin specific primers. Negative images were used to visualize weaker bands. C) CD14 + cells were infected at an MOI = 1 with FIX-WT, FIX-ΔLUNA or FIX-Rev. D–E) Protein was isolated from infected CD14 + cells at the indicated time points and subjected to western blot analysis. Blots were probed with either a rabbit monoclonal a-LUNA or a mouse monoclonal a-IE1 antibody along with a-Tubulin as a loading control. qRT-PCR analysis was used to assay gene expression of UL138, vIL10 and US28 at the indicated times post infection. Viral mRNA was normalized to actin. All samples were tested in triplicate. Abbreviations used: d, days post infection., Mo; mock. IL6 was added at 10 dpi.

    Techniques Used: Infection, Cell Differentiation, Expressing, Synthesized, Amplification, Isolation, Western Blot, Quantitative RT-PCR

    71) Product Images from "Functional, Non-Clonal IgMa-Restricted B Cell Receptor Interactions with the HIV-1 Envelope gp41 Membrane Proximal External Region"

    Article Title: Functional, Non-Clonal IgMa-Restricted B Cell Receptor Interactions with the HIV-1 Envelope gp41 Membrane Proximal External Region

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007215

    Mapping of IgM regions required for allotype-restricted binding to gp41 MPER. A) Surface plasmon resonance analysis of IgM a and IgM b binding to gp41 MPER. Monomeric or F(ab) 2 fragments derived from IgM a and IgM b mAbs were injected over biotinylated (B) gp41 MPER nominal epitope peptide, anchored to a SA-coated sensor chip, as shown in the top left-hand schema, and described in materials and methods . mAbs 17b and 2F5 were run as negative and positive controls for gp41 MPER binding, respectively. Results are representative of two independent experiments. B) V H family usage in gp41 MPER-sorted IgM + B cells from naïve BALB/c mice. Independent 5′ RACE clones (n) were derived from purified BALB/c splenic B cell populations, either unsorted or sorted into gp41 MPER + fractions and subjected to IgM-specific 5′ RACE analysis, as described in Materials and Methods . V H family frequencies within expressed V H repertoires was determined by Ig Blast analysis of sequences. Statistical comparisons between unsorted and gp41 MPER+ fractions were performed using the Fisher Exact test (*, p
    Figure Legend Snippet: Mapping of IgM regions required for allotype-restricted binding to gp41 MPER. A) Surface plasmon resonance analysis of IgM a and IgM b binding to gp41 MPER. Monomeric or F(ab) 2 fragments derived from IgM a and IgM b mAbs were injected over biotinylated (B) gp41 MPER nominal epitope peptide, anchored to a SA-coated sensor chip, as shown in the top left-hand schema, and described in materials and methods . mAbs 17b and 2F5 were run as negative and positive controls for gp41 MPER binding, respectively. Results are representative of two independent experiments. B) V H family usage in gp41 MPER-sorted IgM + B cells from naïve BALB/c mice. Independent 5′ RACE clones (n) were derived from purified BALB/c splenic B cell populations, either unsorted or sorted into gp41 MPER + fractions and subjected to IgM-specific 5′ RACE analysis, as described in Materials and Methods . V H family frequencies within expressed V H repertoires was determined by Ig Blast analysis of sequences. Statistical comparisons between unsorted and gp41 MPER+ fractions were performed using the Fisher Exact test (*, p

    Techniques Used: Binding Assay, SPR Assay, Derivative Assay, Injection, Chromatin Immunoprecipitation, Mouse Assay, Purification

    72) Product Images from "cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions"

    Article Title: cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp514

    Possibilities of disulfide bond formation and the activity of the peptides. ( A ) Two possibilities out of various permutations for the disulfide bond formation were designed and tested. Cys4-2-A denotes clone 2 of S2-Cys4 series containing Type-A disulfide bond pattern i.e. C1 and C4, C2 and C3, while Cys4-2-B denotes clone 2 with disulfide pattern i.e. C1 and C3, C2 and C4. The schematic structures are shown on the right. ( B ) Biochemical binding assay. An in vitro biochemical assay based on was designed as shown in the schematic on the right-hand side. The biotinylated peptides were allowed to bind to IL-6R followed by binding of SA-HRP and detection with the addition of substrate. Non-coated beads denote beads without IL-6R. Cys4-2-A was found to bind to IL-6R and Cys4-2-B could not bind (also shown in the schematic on the right-hand side).
    Figure Legend Snippet: Possibilities of disulfide bond formation and the activity of the peptides. ( A ) Two possibilities out of various permutations for the disulfide bond formation were designed and tested. Cys4-2-A denotes clone 2 of S2-Cys4 series containing Type-A disulfide bond pattern i.e. C1 and C4, C2 and C3, while Cys4-2-B denotes clone 2 with disulfide pattern i.e. C1 and C3, C2 and C4. The schematic structures are shown on the right. ( B ) Biochemical binding assay. An in vitro biochemical assay based on was designed as shown in the schematic on the right-hand side. The biotinylated peptides were allowed to bind to IL-6R followed by binding of SA-HRP and detection with the addition of substrate. Non-coated beads denote beads without IL-6R. Cys4-2-A was found to bind to IL-6R and Cys4-2-B could not bind (also shown in the schematic on the right-hand side).

    Techniques Used: Activity Assay, Binding Assay, In Vitro

    Selection of IL-6R binding molecules from a random library. ( A ) Selection schemes for the enrichment of disulfide bond-containing molecules. The initial library was subjected to affinity selection for three rounds (R1–R3). In Scheme 1 (mRNA display-like procedure), from R4 onward, random mutagenesis was introduced in the PCR amplification step of the R3 selected molecules followed by selection upto R12. In Scheme 2 (cDNA display-like procedure), in addition to random mutagenesis, post-translational modifications were also introduced and the peptides were allowed to fold in the presence of protein disulfide isomerase (PDI) and redox conditions followed by selection upto R9. The displayed peptides were purified owing to the ease of buffer exchange in solid-phase handling. The enriched pools from both the schemes were cloned and sequenced. ( B ) Library construction and sequence characteristics of the selected molecules. A random library was constructed containing 32 residues (shown by X), a fixed cysteine at the N-terminus and 6×-His at the C-terminus for the purification of the peptides. The library was subjected to affinity selection using the two schemes described in (A). Selection with Scheme 1 generated molecules containing two cysteine residues (denoted as Cys-2)—one fixed cysteine and the second from the random region (shown in red). The consensus sequence is shown as S1-Cys2-con and point mutations are highlighted in white. In Scheme 2, molecules containing two (Cys-2; top) and four cysteine residues (Cys-4; bottom) were enriched. S2-Cys2-con and S2-Cys4-con are the consensus sequences of Cys2 and Cys4 peptides, respectively. Point mutations are highlighted in white.
    Figure Legend Snippet: Selection of IL-6R binding molecules from a random library. ( A ) Selection schemes for the enrichment of disulfide bond-containing molecules. The initial library was subjected to affinity selection for three rounds (R1–R3). In Scheme 1 (mRNA display-like procedure), from R4 onward, random mutagenesis was introduced in the PCR amplification step of the R3 selected molecules followed by selection upto R12. In Scheme 2 (cDNA display-like procedure), in addition to random mutagenesis, post-translational modifications were also introduced and the peptides were allowed to fold in the presence of protein disulfide isomerase (PDI) and redox conditions followed by selection upto R9. The displayed peptides were purified owing to the ease of buffer exchange in solid-phase handling. The enriched pools from both the schemes were cloned and sequenced. ( B ) Library construction and sequence characteristics of the selected molecules. A random library was constructed containing 32 residues (shown by X), a fixed cysteine at the N-terminus and 6×-His at the C-terminus for the purification of the peptides. The library was subjected to affinity selection using the two schemes described in (A). Selection with Scheme 1 generated molecules containing two cysteine residues (denoted as Cys-2)—one fixed cysteine and the second from the random region (shown in red). The consensus sequence is shown as S1-Cys2-con and point mutations are highlighted in white. In Scheme 2, molecules containing two (Cys-2; top) and four cysteine residues (Cys-4; bottom) were enriched. S2-Cys2-con and S2-Cys4-con are the consensus sequences of Cys2 and Cys4 peptides, respectively. Point mutations are highlighted in white.

    Techniques Used: Selection, Binding Assay, Mutagenesis, Polymerase Chain Reaction, Amplification, Purification, Buffer Exchange, Clone Assay, Sequencing, Construct, Generated

    73) Product Images from "Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput sequencing method"

    Article Title: Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput sequencing method

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq107

    Schematic of the ultra HTS strategy used to measure RNA editing. ( A ) A representative full-sequence read with the five edited sites labeled. Guanosines at edited sites correspond to inosines in the original RNA transcript from which the sequenced DNA is derived. ( B ) Ultra HTS sequencing produced 22 652 442/122 828 564 sequences. Non-edited region 1 and non-edited region 2 were used to filter sequences with misreads in those regions (13 790 980 sequences). Non-edited regions 3, 4 and 5 were then used to filter the remaining 8 861 462/92 958 400 sequences in a similar fashion to remove the sequences containing misreads in those regions, and theoretically impossible transcripts (in other words, those with a C or T at an edited site) were also filtered (213 997/21 770 405 sequences failed after applying the last two filters), leaving 8 647 465/71 187 995 sequences used for subsequent analysis.
    Figure Legend Snippet: Schematic of the ultra HTS strategy used to measure RNA editing. ( A ) A representative full-sequence read with the five edited sites labeled. Guanosines at edited sites correspond to inosines in the original RNA transcript from which the sequenced DNA is derived. ( B ) Ultra HTS sequencing produced 22 652 442/122 828 564 sequences. Non-edited region 1 and non-edited region 2 were used to filter sequences with misreads in those regions (13 790 980 sequences). Non-edited regions 3, 4 and 5 were then used to filter the remaining 8 861 462/92 958 400 sequences in a similar fashion to remove the sequences containing misreads in those regions, and theoretically impossible transcripts (in other words, those with a C or T at an edited site) were also filtered (213 997/21 770 405 sequences failed after applying the last two filters), leaving 8 647 465/71 187 995 sequences used for subsequent analysis.

    Techniques Used: Sequencing, Labeling, Derivative Assay, Produced

    74) Product Images from "Flavonoids activate pregnane x receptor-mediated CYP3A4 gene expression by inhibiting cyclin-dependent kinases in HepG2 liver carcinoma cells"

    Article Title: Flavonoids activate pregnane x receptor-mediated CYP3A4 gene expression by inhibiting cyclin-dependent kinases in HepG2 liver carcinoma cells

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-11-23

    Cdk5 activation attenuates PXR-mediated gene expression . (A) Overexpression of Cdk5 reduces PXR-mediated CYP3A4 promoter activity. HepG2 was transiently co-transfected with indicated plasmids as well as CMV- renilla luciferase plasmid (as a transfection control). In cases wherein Cdk5, FLAG-PXR, or CYP3A4-luc constructs were not used, a pcDNA3 vector was used instead. Equal amount of each plasmid (for a total of 1 μg combined) was used to transfect 8 × 10 5 cells seeded in 6-well plates. Cells were treated with 5 μM rifampicin or DMSO for 24 h after transfection before Dual-glo luciferase assay. CYP3A4 promoter activity (expressed as relative luciferase unit, or RLU) was normalized by using activity of the CMV- renilla . The values represent the average of 8 independent experiments, with the standard deviation denoted as bars. The significance of the difference between datasets was determined by using the Student's t test. (B) Expression of PXR and Cdk5. Transfections were performed as described in (A). Western blotting shown was from a representative experiment. (C) Downregulation of Cdk5 enhances the activity of PXR. HepG2 was transfected with siRNA specific for Cdk5 (siRNA-Cdk5) or control siRNA (siRNA-Control) in addition to indicated plasmids and CMV- renilla as described in Methods. Cells were treated with 5 μM rifampicin. CYP3A4 promoter activity was expressed as RLU as described in (A). The values represent the average of 6 independent experiments. The efficiency of Cdk5 knockdown was verified in (D).
    Figure Legend Snippet: Cdk5 activation attenuates PXR-mediated gene expression . (A) Overexpression of Cdk5 reduces PXR-mediated CYP3A4 promoter activity. HepG2 was transiently co-transfected with indicated plasmids as well as CMV- renilla luciferase plasmid (as a transfection control). In cases wherein Cdk5, FLAG-PXR, or CYP3A4-luc constructs were not used, a pcDNA3 vector was used instead. Equal amount of each plasmid (for a total of 1 μg combined) was used to transfect 8 × 10 5 cells seeded in 6-well plates. Cells were treated with 5 μM rifampicin or DMSO for 24 h after transfection before Dual-glo luciferase assay. CYP3A4 promoter activity (expressed as relative luciferase unit, or RLU) was normalized by using activity of the CMV- renilla . The values represent the average of 8 independent experiments, with the standard deviation denoted as bars. The significance of the difference between datasets was determined by using the Student's t test. (B) Expression of PXR and Cdk5. Transfections were performed as described in (A). Western blotting shown was from a representative experiment. (C) Downregulation of Cdk5 enhances the activity of PXR. HepG2 was transfected with siRNA specific for Cdk5 (siRNA-Cdk5) or control siRNA (siRNA-Control) in addition to indicated plasmids and CMV- renilla as described in Methods. Cells were treated with 5 μM rifampicin. CYP3A4 promoter activity was expressed as RLU as described in (A). The values represent the average of 6 independent experiments. The efficiency of Cdk5 knockdown was verified in (D).

    Techniques Used: Activation Assay, Expressing, Over Expression, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Construct, Standard Deviation, Western Blot

    75) Product Images from "Fitness and Phenotypic Characterization of Miltefosine-Resistant Leishmania major"

    Article Title: Fitness and Phenotypic Characterization of Miltefosine-Resistant Leishmania major

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003948

    Growth curves of L . major WT and MIL-resistant promastigotes growing in the presence of 30μM MIL or absence of MIL selection. Log-phase promastigotes cultures were counted daily until they reached stationary phase. Concentration was determined microscopically by counting in a Neubauer chamber. Results are the average of triplicate experiments ± SD.
    Figure Legend Snippet: Growth curves of L . major WT and MIL-resistant promastigotes growing in the presence of 30μM MIL or absence of MIL selection. Log-phase promastigotes cultures were counted daily until they reached stationary phase. Concentration was determined microscopically by counting in a Neubauer chamber. Results are the average of triplicate experiments ± SD.

    Techniques Used: Selection, Concentration Assay

    Metacyclogenesis in WT and MIL-resistant L . major . L . major promastigotes resistant to MIL exhibit increased metacyclogenesis as determined by qRT-PCR of SHERP expression relative to housekeeping gene GAPDH and normalized to WT expression levels (left). 5-day stationary parasites were subjected to peanut agglutination and Ficoll-400 gradients and percentage of metacyclics is shown (right). Results are the average of triplicate experiments ± SD. Statistical differences determined with a Student’s t test relative to control values (* p
    Figure Legend Snippet: Metacyclogenesis in WT and MIL-resistant L . major . L . major promastigotes resistant to MIL exhibit increased metacyclogenesis as determined by qRT-PCR of SHERP expression relative to housekeeping gene GAPDH and normalized to WT expression levels (left). 5-day stationary parasites were subjected to peanut agglutination and Ficoll-400 gradients and percentage of metacyclics is shown (right). Results are the average of triplicate experiments ± SD. Statistical differences determined with a Student’s t test relative to control values (* p

    Techniques Used: Quantitative RT-PCR, Expressing, Agglutination

    Virulence of WT and MIL-resistant L . major . R40 demonstrate attenuated virulence in vivo compared with WT promastigotes. 1×10 6 WT (n = 5) and R40 (n = 6) metacyclic promastigotes were injected into the footpads of female BALB/c mice. Lesion size was recorded weekly by taking measurements of footpad thickness with a Vernier caliper, results are averages ± SD.
    Figure Legend Snippet: Virulence of WT and MIL-resistant L . major . R40 demonstrate attenuated virulence in vivo compared with WT promastigotes. 1×10 6 WT (n = 5) and R40 (n = 6) metacyclic promastigotes were injected into the footpads of female BALB/c mice. Lesion size was recorded weekly by taking measurements of footpad thickness with a Vernier caliper, results are averages ± SD.

    Techniques Used: In Vivo, Injection, Mouse Assay

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    Reverse Transcription Polymerase Chain Reaction:

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    Generated:

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    Polymerase Chain Reaction:

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    Binding Assay:

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    Mutagenesis:

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    Isolation:

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    Purification:

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    Sequencing:

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    Quantitative RT-PCR:

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    IA:

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    Mouse Assay:

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    Plasmid Preparation:

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    Software:

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    Real-time Polymerase Chain Reaction:

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    Article Title: KDELR2 Competes with Measles Virus Envelope Proteins for Cellular Chaperones Reducing Their Chaperone-Mediated Cell Surface Transport
    Article Snippet: Paragraph title: 2.6. Real Time qPCR ... Isolated RNA was reverse transcribed in cDNA using the RevertAid first strand cDNA synthesis kit (Fermentas).

    Article Title: Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability
    Article Snippet: .. RT-qPCR quantification of RNA abundance 2–50 ng of mRNA (depending on sample) was used for reverse transcription using Superscript II [Life technologies] with random hexamer primers. cDNA was quantified on a StepOnePlus Real-Time PCR system using a SYBR green PCR mix [ThermoFisher] with gene specific primers ( ). .. High-throughput sequencing and RNAseq quantification 50–100 ng mRNA was used as input material to generate strand-specific sequencing libraries using the NEXTflex Rapid Directional Illumina RNA-Seq Library Prep Kit [BioO] according to the manufacturer’s instructions.

    Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template
    Article Snippet: .. Quantitative PCR Forty-eight hours post-transfection, cells were spun down at 500 × g for 5 min. Genomic DNA was extracted and purified from cell pellets using Purelink genomic DNA purification kit (Invitrogen). .. In triplicate, SYBR-based qPCR reactions were performed using PowerUp SYBR Green Master Mix (Applied Biosciences) on a Bio-Rad CFX96 real-time thermocycler.

    Article Title: 25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes
    Article Snippet: Briefly, extraction of total RNA from transfected or untransfected Caco2 cells was performed 72 h after transfection using TRIzol Reagent (Applied Biosystems, Monza, Italy). .. Quantitative RT-PCR was performed with 30 ng of cDNA using TaqMan Gene Expression Assay kits for OSBP and β-actin, TaqMan Fast Universal PCR Master Mix, and 7500 Fast Real-Time PCR System (Applied Biosystems).

    Ethanol Precipitation:

    Article Title: Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5′UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication
    Article Snippet: Genomic HCV RNA was synthesized using the MEGAscript T7 kit (Thermo Fisher Scientific) with 1 µg of Xba I-linearized, mung bean nuclease-treated pER-1b or pER-1b containing 5′UTR or NS5B mutations (Thermo Fisher Scientific). .. RNA transcription reactions proceeded for 4 to 6 h at 37 °C, followed by DNA template removal using RNase-free DNase (Thermo Fisher Scientific), organic extraction and ethanol precipitation.

    Spectrophotometry:

    Article Title: Negative Cellular Effects of Urban Particulate Matter on Human Keratinocytes Are Mediated by P38 MAPK and NF-κB-dependent Expression of TRPV 1
    Article Snippet: Reverse Transcription HaCaT cells were grown in serum-free culture medium, with or without the indicated concentrations of UPM, for two days, and the total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. .. The purified RNA was then treated with DNase (Ambion, Austin, TX, USA) and analyzed using an Agilent Bioanalyzer (Agilent Technologies, Waldbronn, Germany) and a NanoDrop 8000 spectrophotometer (Thermo Scientific, Schwerte, Germany) to measure the RNA concentration, integrity, and purity.

    Article Title: 25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes
    Article Snippet: Briefly, extraction of total RNA from transfected or untransfected Caco2 cells was performed 72 h after transfection using TRIzol Reagent (Applied Biosystems, Monza, Italy). .. Concentration and purity of the extracted RNA were assessed by spectrophotometry (A260/A280).

    Produced:

    Article Title: Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5′UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication
    Article Snippet: HCV Production and Immunofluorescence Infectious virus was produced in Huh-7.5 cells as outlined by Wakita et al 2005 [ ]. .. Genomic HCV RNA was synthesized using the MEGAscript T7 kit (Thermo Fisher Scientific) with 1 µg of Xba I-linearized, mung bean nuclease-treated pER-1b or pER-1b containing 5′UTR or NS5B mutations (Thermo Fisher Scientific).

    Concentration Assay:

    Article Title: Negative Cellular Effects of Urban Particulate Matter on Human Keratinocytes Are Mediated by P38 MAPK and NF-κB-dependent Expression of TRPV 1
    Article Snippet: Reverse Transcription HaCaT cells were grown in serum-free culture medium, with or without the indicated concentrations of UPM, for two days, and the total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. .. The purified RNA was then treated with DNase (Ambion, Austin, TX, USA) and analyzed using an Agilent Bioanalyzer (Agilent Technologies, Waldbronn, Germany) and a NanoDrop 8000 spectrophotometer (Thermo Scientific, Schwerte, Germany) to measure the RNA concentration, integrity, and purity.

    Article Title: 25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes
    Article Snippet: Briefly, extraction of total RNA from transfected or untransfected Caco2 cells was performed 72 h after transfection using TRIzol Reagent (Applied Biosystems, Monza, Italy). .. Concentration and purity of the extracted RNA were assessed by spectrophotometry (A260/A280).

    DNA Purification:

    Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template
    Article Snippet: .. Quantitative PCR Forty-eight hours post-transfection, cells were spun down at 500 × g for 5 min. Genomic DNA was extracted and purified from cell pellets using Purelink genomic DNA purification kit (Invitrogen). .. In triplicate, SYBR-based qPCR reactions were performed using PowerUp SYBR Green Master Mix (Applied Biosciences) on a Bio-Rad CFX96 real-time thermocycler.

    High Throughput Screening Assay:

    Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
    Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

    Variant Assay:

    Article Title: A Genome-Wide Scan for MicroRNA-Related Genetic Variants Associated With Primary Open-Angle Glaucoma
    Article Snippet: In addition, this experiment was used to determine the impact of candidate variant on the miRNA–target gene interaction. .. HEK293 cells (n = 10,000) were plated into 96-well plates and cotransfected with 1 μg pGL3 containing the 3′UTR with either the major or minor allele, miRNA mimic (mirVana Mimics; Thermo Fischer Scientific, Waltham, MA, USA), and a plasmid expressing the Renilla luciferase that served as transfection control, with Lipofectamine RNAiMAX (Invitrogen).

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    Thermo Fisher turbo dna free kit
    Active P-TEFb Is Vital for the Pol II Transcriptional Response to Genotoxic Stress (A) (Top) Schematic depicting major steps in the generation of 4sU-labeled transcripts (4sU RNA) for 4sU-seq. (Bottom) Pie charts showing the fractions of DE protein-coding genes (mRNA) in 4-NQO-treated HeLa cells as assessed by 4sU-seq (n = 2). (B) Bar charts showing the number of DE classes of transcripts in HeLa cells as assessed by 4sU-seq (n = 2). The degrees of differential expression are presented according to the legend. Conditions with (in hours) and without (−) 4-NQO or FP are shown. (C) Boxplots indicating the distribution of gene lengths for upregulated and downregulated protein-coding genes. Median gene length for each group is shown. (D) Top Molecular and Cellular Functions categories of the 4FP gene set as identified by IPA. The number of affected genes per category is shown on the right. (E) RT-qPCR of the indicated <t>DNA</t> damage-induced unspliced (pre-mRNA), uaRNA, and eRNA transcripts. HeLa cells were treated as indicated by the legend. Results were normalized to the DMSO control and are presented as the mean ± SEM (n = 3). ∗ p
    Turbo Dna Free Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dna free dna removal kit
    CX-5461 treatment disrupts HCMV replication at the stage of viral <t>DNA</t> synthesis (A) MRC-5 fibroblasts were infected with TB40/E virus or mock infected at an MOI of 3 IU/cell. Cells were treated with 0.5 μM CX-5461 or vehicle control at 2 or 48 hpi as indicated. Total <t>RNA</t> was isolated at the indicated times and IE1 and GAPDH levels were determined by quantitative RT-PCR. (B) Under the same conditions, viral DNA levels were determined using quantitative PCR and primers against UL123 and GAPDH. (C) Fibroblasts were infected as above and treated starting at 48 hpi. Whole cell lysates were collected at multiple times post infection and analyzed by Western blot using antibodies against the indicated proteins. (D) Samples were fixed 96 hpi and stained using the indicated antibody. Arrows identify two examples of disrupted replication compartments. Quantitative PCR data are from three biological replicates and two technical replicates with error bars representing standard deviation from the mean.
    Dna Free Dna Removal Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dna free rna
    Detection of yeast and hyphal growth condition-specific RNAs initiated upstream of ALS3 in strain SC5314. (A) Schematic diagram of the <t>DNA</t> upstream of ALS3 showing the location of the ALS3-Y initiation site during yeast conditions and the primers and probe used for detection. The numbers are relative to the ALS3 translation start site (denoted +1). (B) Detection of ALS3-Y by strand specific PCR. (C) Northern blot for detection of ALS3-Y. <t>RNA</t> from strain SC5314 was probed with DNA fragment A1 (hatched rectangle in (A)) spanning the region from -431 to -80 bp upstream of the ALS3 translation start site. The membrane was exposed for three weeks. (D) qRT-PCR showing increased abundance of ALS3-Y under yeast growth conditions compared to hyphal growth conditions. Error bars represent the standard deviation from the mean. The fold change in the amount of the ALS3-Y transcript and hyphal conditions was significant as determined by the Student’s t test (*indicates P
    Dna Free Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Active P-TEFb Is Vital for the Pol II Transcriptional Response to Genotoxic Stress (A) (Top) Schematic depicting major steps in the generation of 4sU-labeled transcripts (4sU RNA) for 4sU-seq. (Bottom) Pie charts showing the fractions of DE protein-coding genes (mRNA) in 4-NQO-treated HeLa cells as assessed by 4sU-seq (n = 2). (B) Bar charts showing the number of DE classes of transcripts in HeLa cells as assessed by 4sU-seq (n = 2). The degrees of differential expression are presented according to the legend. Conditions with (in hours) and without (−) 4-NQO or FP are shown. (C) Boxplots indicating the distribution of gene lengths for upregulated and downregulated protein-coding genes. Median gene length for each group is shown. (D) Top Molecular and Cellular Functions categories of the 4FP gene set as identified by IPA. The number of affected genes per category is shown on the right. (E) RT-qPCR of the indicated DNA damage-induced unspliced (pre-mRNA), uaRNA, and eRNA transcripts. HeLa cells were treated as indicated by the legend. Results were normalized to the DMSO control and are presented as the mean ± SEM (n = 3). ∗ p

    Journal: Molecular Cell

    Article Title: P-TEFb Activation by RBM7 Shapes a Pro-survival Transcriptional Response to Genotoxic Stress

    doi: 10.1016/j.molcel.2019.01.033

    Figure Lengend Snippet: Active P-TEFb Is Vital for the Pol II Transcriptional Response to Genotoxic Stress (A) (Top) Schematic depicting major steps in the generation of 4sU-labeled transcripts (4sU RNA) for 4sU-seq. (Bottom) Pie charts showing the fractions of DE protein-coding genes (mRNA) in 4-NQO-treated HeLa cells as assessed by 4sU-seq (n = 2). (B) Bar charts showing the number of DE classes of transcripts in HeLa cells as assessed by 4sU-seq (n = 2). The degrees of differential expression are presented according to the legend. Conditions with (in hours) and without (−) 4-NQO or FP are shown. (C) Boxplots indicating the distribution of gene lengths for upregulated and downregulated protein-coding genes. Median gene length for each group is shown. (D) Top Molecular and Cellular Functions categories of the 4FP gene set as identified by IPA. The number of affected genes per category is shown on the right. (E) RT-qPCR of the indicated DNA damage-induced unspliced (pre-mRNA), uaRNA, and eRNA transcripts. HeLa cells were treated as indicated by the legend. Results were normalized to the DMSO control and are presented as the mean ± SEM (n = 3). ∗ p

    Article Snippet: RNA samples were DNase-treated with the Turbo DNA-Free kit (Thermo Fisher Scientific), reverse transcribed with SuperScript III reverse transcriptase (Thermo Fisher Sceintific) and random hexamers (Thermo Fisher Scientific), and amplified using FastStart Universal SYBR Green QPCR Master (Rox) (Sigma), RNA-specific primer pair, and Stratagene Mx3005 qPCR machine.

    Techniques: Labeling, Expressing, Indirect Immunoperoxidase Assay, Quantitative RT-PCR

    GRF9 directly regulates ORG3 . (A) GRF9 DNA-binding sequences determined using in vitro binding site selection. All selected oligonucleotides contain a functional GRF9-binding site as verified in DNA-binding assays. The nucleotides in the core binding sequence are shown in red. The logo of the GRF9 binding sequence profile was generated using the MEME program ( http://meme.sdsc.edu/meme/cgi-bin/meme.cgi ). (B) GRF9 binds in vitro to the ORG3 fragment harboring the GRF9 binding site. A schematic view of the ORG3 promoter (around 1.5 kb upstream of the translation start site) containing the GRF9 binding sequence (BS) is shown at the bottom (BS; black box). Electrophoretic mobility shift assay (EMSA) using GRF9-CELD protein and a 40-bp sequence of the ORG3 promoter harboring the GRF9 BS. GRF9-CELD protein incubated with those oligonucleotides causes retardation ('Band shift'). Retardation disappeared in the presence of competitor (unlabeled probe at high concentration) whilst adding a molar excess of mutated probe did not block the interaction between GRF9 protein and the labelled probe, indicating specific binding of GRF9 to the CTGACA binding site. (C) Laser scanning confocal microscopy images showing nuclear localization of GRF9-GFP fusion protein in 3-week-old transgenic Arabidopsis plants expressing GFP-tagged GRF9 protein. (D) Expression level of GRF9 and ORG3 in Pro 35S : GRF9-GFP and WT (Col-0) plants. Expression was determined by qRT-PCR and values represent the means of replicates from three biological replicates ± SD. (E) GRF9 binds in vivo to the ORG3 promoter. ChIP-qPCR results of 5-day-old Pro 35S : GRF9-GFP Arabidopsis seedlings. Data represent average enrichment (fold change, FC) in three independent biological replicates ± SD. (F) GRF9 transactivates the ORG3 promoter in vivo . Relative luciferase activity detected in Arabidopsis mesophyll cell protoplasts. Data are means ± SD of three independent transformations, each representing five technical replicates. The asterisks indicate significant difference (Student's t -test; p

    Journal: PLoS Genetics

    Article Title: GROWTH-REGULATING FACTOR 9 negatively regulates arabidopsis leaf growth by controlling ORG3 and restricting cell proliferation in leaf primordia

    doi: 10.1371/journal.pgen.1007484

    Figure Lengend Snippet: GRF9 directly regulates ORG3 . (A) GRF9 DNA-binding sequences determined using in vitro binding site selection. All selected oligonucleotides contain a functional GRF9-binding site as verified in DNA-binding assays. The nucleotides in the core binding sequence are shown in red. The logo of the GRF9 binding sequence profile was generated using the MEME program ( http://meme.sdsc.edu/meme/cgi-bin/meme.cgi ). (B) GRF9 binds in vitro to the ORG3 fragment harboring the GRF9 binding site. A schematic view of the ORG3 promoter (around 1.5 kb upstream of the translation start site) containing the GRF9 binding sequence (BS) is shown at the bottom (BS; black box). Electrophoretic mobility shift assay (EMSA) using GRF9-CELD protein and a 40-bp sequence of the ORG3 promoter harboring the GRF9 BS. GRF9-CELD protein incubated with those oligonucleotides causes retardation ('Band shift'). Retardation disappeared in the presence of competitor (unlabeled probe at high concentration) whilst adding a molar excess of mutated probe did not block the interaction between GRF9 protein and the labelled probe, indicating specific binding of GRF9 to the CTGACA binding site. (C) Laser scanning confocal microscopy images showing nuclear localization of GRF9-GFP fusion protein in 3-week-old transgenic Arabidopsis plants expressing GFP-tagged GRF9 protein. (D) Expression level of GRF9 and ORG3 in Pro 35S : GRF9-GFP and WT (Col-0) plants. Expression was determined by qRT-PCR and values represent the means of replicates from three biological replicates ± SD. (E) GRF9 binds in vivo to the ORG3 promoter. ChIP-qPCR results of 5-day-old Pro 35S : GRF9-GFP Arabidopsis seedlings. Data represent average enrichment (fold change, FC) in three independent biological replicates ± SD. (F) GRF9 transactivates the ORG3 promoter in vivo . Relative luciferase activity detected in Arabidopsis mesophyll cell protoplasts. Data are means ± SD of three independent transformations, each representing five technical replicates. The asterisks indicate significant difference (Student's t -test; p

    Article Snippet: Semi-quantitative RT-PCR Total RNA was extracted from ORG3 transgenic and WT plants and genomic DNA contamination was removed using Turbo DNA Free Kit (Ambion/Thermo Fisher Scientific, Darmstadt, Germany). cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific).

    Techniques: Binding Assay, In Vitro, Selection, Functional Assay, Sequencing, Generated, Electrophoretic Mobility Shift Assay, Incubation, Concentration Assay, Blocking Assay, Confocal Microscopy, Transgenic Assay, Expressing, Quantitative RT-PCR, In Vivo, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay

    CX-5461 treatment disrupts HCMV replication at the stage of viral DNA synthesis (A) MRC-5 fibroblasts were infected with TB40/E virus or mock infected at an MOI of 3 IU/cell. Cells were treated with 0.5 μM CX-5461 or vehicle control at 2 or 48 hpi as indicated. Total RNA was isolated at the indicated times and IE1 and GAPDH levels were determined by quantitative RT-PCR. (B) Under the same conditions, viral DNA levels were determined using quantitative PCR and primers against UL123 and GAPDH. (C) Fibroblasts were infected as above and treated starting at 48 hpi. Whole cell lysates were collected at multiple times post infection and analyzed by Western blot using antibodies against the indicated proteins. (D) Samples were fixed 96 hpi and stained using the indicated antibody. Arrows identify two examples of disrupted replication compartments. Quantitative PCR data are from three biological replicates and two technical replicates with error bars representing standard deviation from the mean.

    Journal: Antiviral research

    Article Title: Impact of RNA polymerase I inhibitor CX-5461 on viral kinase-dependent and -independent cytomegalovirus replication

    doi: 10.1016/j.antiviral.2018.02.014

    Figure Lengend Snippet: CX-5461 treatment disrupts HCMV replication at the stage of viral DNA synthesis (A) MRC-5 fibroblasts were infected with TB40/E virus or mock infected at an MOI of 3 IU/cell. Cells were treated with 0.5 μM CX-5461 or vehicle control at 2 or 48 hpi as indicated. Total RNA was isolated at the indicated times and IE1 and GAPDH levels were determined by quantitative RT-PCR. (B) Under the same conditions, viral DNA levels were determined using quantitative PCR and primers against UL123 and GAPDH. (C) Fibroblasts were infected as above and treated starting at 48 hpi. Whole cell lysates were collected at multiple times post infection and analyzed by Western blot using antibodies against the indicated proteins. (D) Samples were fixed 96 hpi and stained using the indicated antibody. Arrows identify two examples of disrupted replication compartments. Quantitative PCR data are from three biological replicates and two technical replicates with error bars representing standard deviation from the mean.

    Article Snippet: Approximately 2 μg of RNA was treated with DNA-free DNA Removal Kit (ThermoFisher Scientific) and used to synthesize cDNA with random hexamers and Superscript III Reverse Transcriptase (ThermoFisher Scientific). qPCR was performed as described using primers against CMV UL123, GAPDH and pre-rRNA external transcribed spacer 1 (ETS) (5′-GAACGGTGGTGTGTCGTTC-3′ and 5′-GCGTCTCGTCTCGTCTCACT-3′).

    Techniques: DNA Synthesis, Infection, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot, Staining, Standard Deviation

    Detection of yeast and hyphal growth condition-specific RNAs initiated upstream of ALS3 in strain SC5314. (A) Schematic diagram of the DNA upstream of ALS3 showing the location of the ALS3-Y initiation site during yeast conditions and the primers and probe used for detection. The numbers are relative to the ALS3 translation start site (denoted +1). (B) Detection of ALS3-Y by strand specific PCR. (C) Northern blot for detection of ALS3-Y. RNA from strain SC5314 was probed with DNA fragment A1 (hatched rectangle in (A)) spanning the region from -431 to -80 bp upstream of the ALS3 translation start site. The membrane was exposed for three weeks. (D) qRT-PCR showing increased abundance of ALS3-Y under yeast growth conditions compared to hyphal growth conditions. Error bars represent the standard deviation from the mean. The fold change in the amount of the ALS3-Y transcript and hyphal conditions was significant as determined by the Student’s t test (*indicates P

    Journal: PLoS ONE

    Article Title: Release of transcriptional repression through the HCR promoter region confers uniform expression of HWP1 on surfaces of Candida albicans germ tubes

    doi: 10.1371/journal.pone.0192260

    Figure Lengend Snippet: Detection of yeast and hyphal growth condition-specific RNAs initiated upstream of ALS3 in strain SC5314. (A) Schematic diagram of the DNA upstream of ALS3 showing the location of the ALS3-Y initiation site during yeast conditions and the primers and probe used for detection. The numbers are relative to the ALS3 translation start site (denoted +1). (B) Detection of ALS3-Y by strand specific PCR. (C) Northern blot for detection of ALS3-Y. RNA from strain SC5314 was probed with DNA fragment A1 (hatched rectangle in (A)) spanning the region from -431 to -80 bp upstream of the ALS3 translation start site. The membrane was exposed for three weeks. (D) qRT-PCR showing increased abundance of ALS3-Y under yeast growth conditions compared to hyphal growth conditions. Error bars represent the standard deviation from the mean. The fold change in the amount of the ALS3-Y transcript and hyphal conditions was significant as determined by the Student’s t test (*indicates P

    Article Snippet: Briefly, DNA-free RNA from cells grown under yeast conditions was prepared by treating total RNA from strain HCRc, HCRc-TerN or HCRc-TerC with DNase I (ThermoFisher Scientific, Carlsbad, CA) for 1 h at 37°C.

    Techniques: Polymerase Chain Reaction, Northern Blot, Quantitative RT-PCR, Standard Deviation

    Detection of an mRNA isoform initiating upstream of the HWP1 transcription start site under hyphal growth conditions. (A) Schematic diagram of the region upstream of HWP1 showing the locations of the HWP1-H initiation site and the primers and probe used for its detection. The numbers are relative to the HWP1 transcription initiation site (denoted +1), which is 57 bp upstream from the translation start site [ 23 ]. Transcripts are indicated with dotted lines. (B) Detection of HWP-H by strand-specific RT-PCR. (C) Northern blot prepared using total RNA from strains SKD14 ( hwp1∆/hwp1∆) , CAI4 ( HWP1/HWP1 ) and SKD4 (hcr∆/hcr∆) probed with H41 (hatched rectangle in (A)) to detect HWP1-H and pBS+13 [ 23 ] to detect HWP1 . HWP1-H was detected under hyphal growth conditions in the wild-type strain but not in the hcr∆/hcr∆ or hwp1∆/hwp1∆ strains. (D) Localization of the HWP1-H initiation site by 5’ RACE. The first PCR resulted in a product derived from mature HWP1 mRNA (arrow). The second PCR reaction generated a PCR product derived from HWP1-H (arrow). The initiation site was found to be 836 bp upstream of the HWP1 transcription start site by DNA sequencing. HWP1-H was not detected under yeast growth conditions or in a strain in which the entire HWP1 locus was deleted.

    Journal: PLoS ONE

    Article Title: Release of transcriptional repression through the HCR promoter region confers uniform expression of HWP1 on surfaces of Candida albicans germ tubes

    doi: 10.1371/journal.pone.0192260

    Figure Lengend Snippet: Detection of an mRNA isoform initiating upstream of the HWP1 transcription start site under hyphal growth conditions. (A) Schematic diagram of the region upstream of HWP1 showing the locations of the HWP1-H initiation site and the primers and probe used for its detection. The numbers are relative to the HWP1 transcription initiation site (denoted +1), which is 57 bp upstream from the translation start site [ 23 ]. Transcripts are indicated with dotted lines. (B) Detection of HWP-H by strand-specific RT-PCR. (C) Northern blot prepared using total RNA from strains SKD14 ( hwp1∆/hwp1∆) , CAI4 ( HWP1/HWP1 ) and SKD4 (hcr∆/hcr∆) probed with H41 (hatched rectangle in (A)) to detect HWP1-H and pBS+13 [ 23 ] to detect HWP1 . HWP1-H was detected under hyphal growth conditions in the wild-type strain but not in the hcr∆/hcr∆ or hwp1∆/hwp1∆ strains. (D) Localization of the HWP1-H initiation site by 5’ RACE. The first PCR resulted in a product derived from mature HWP1 mRNA (arrow). The second PCR reaction generated a PCR product derived from HWP1-H (arrow). The initiation site was found to be 836 bp upstream of the HWP1 transcription start site by DNA sequencing. HWP1-H was not detected under yeast growth conditions or in a strain in which the entire HWP1 locus was deleted.

    Article Snippet: Briefly, DNA-free RNA from cells grown under yeast conditions was prepared by treating total RNA from strain HCRc, HCRc-TerN or HCRc-TerC with DNase I (ThermoFisher Scientific, Carlsbad, CA) for 1 h at 37°C.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Northern Blot, Polymerase Chain Reaction, Derivative Assay, Generated, DNA Sequencing